Methods of treating cancer

ABSTRACT

This invention relates to methods of treating cancer in a subject in need thereof, e.g., in a human in need thereof, comprising determining at least one of the following in a sample from said human: (a) the presence or absence of a mutation at the alanine 677 (A677) residue in EZH2 in a sample from said human; or (b) the presence or absence of a mutation at the tyrosine 641 (Y641) residue in EZH2; or (c) the presence or absence of an increased level of H3K27me3 as compared to a control, and administering to said human an effective amount of an EZH2 inhibitor or a pharmaceutically acceptable salt thereof if at least one of said A677 mutation, Y641 mutation, or increased level of H3K27me3 is present in said sample

This application is a divisional of U.S. application Ser. No. 14/346,359filed on Mar. 21, 2014, which is a 371 of InternationalPCT/US2012/058188 filed on Sep. 30, 2012, which claims the benefit ofU.S. Provisional Application 61/541,304 filed on Sep. 30, 2011, all ofwhich are incorporated herein in their entirety.

FIELD

This invention relates to methods of treating cancer in a subject inneed thereof.

BACKGROUND

The expanding development and use of targeted therapies for cancertreatment reflects an increasing understanding of key oncogenicpathways, and how the targeted perturbation of these pathwayscorresponds to clinical response. Difficulties in predicting efficacy totargeted therapies is likely a consequence of the limited globalknowledge of causal mechanisms for pathway deregulation (e.g. activatingmutations, amplifications). Pre-clinical translational research studiesfor oncology therapies focuses on determining what tumor type andgenotypes are most likely to benefit from treatment. Treating selectedpatient populations may help maximize the potential of a therapy.Pre-clinical cellular response profiling of tumor models has become acornerstone in development of novel cancer therapeutics. Efforts topredict clinical efficacy using cohorts of in vitro tumor models havebeen successful (e.g. EGFR inhibitors are selectively useful in thosetumors harboring EGFR mutations). Thus, expansive panels of diversetumor derived cell lines could recapitulate an ‘all comers’ efficacytrial; thereby identifying which histologies and specific tumorgenotypes are most likely to benefit from treatment. Numerous specificmolecular markers are now used to identify patients most likely tobenefit in a clinical setting.

EZH2 (enhancer of zeste homolog 2; human EZH2 gene: Cardoso, C, et al;European J of Human Genetics, Vol. 8, No. 3 Pages 174-180, 2000) is thecatalytic subunit of the Polycomb Repressor Complex 2 (PRC2) whichfunctions to silence target genes by tri-methylating lysine 27 ofhistone H3 (H3K27me3). Histone H3 is one of the five main histoneproteins involved in the structure of chromatin in eukaryotic cells.Featuring a main globular domain and a long N-terminal tail, Histonesare involved with the structure of the nucleosomes, a ‘beads on astring’ structure. Histone proteins are highly post-translationallymodified however Histone H3 is the most extensively modified of the fivehistones. The term “Histone H3” alone is purposely ambiguous in that itdoes not distinguish between sequence variants or modification state.Histone H3 is an important protein in the emerging field of epigenetics,where its sequence variants and variable modification states are thoughtto play a role in the dynamic and long term regulation of genes.

EZH2 inhibitors that are useful in treating cancer have been reported inPCT applications PCT/US2011/035336, PCT/US2011/035340, andPCT/US2011/035344, which are incorporated by reference herein. It isdesirable to identify genotypes that are more likely to respond to thesecompounds.

SUMMARY OF THE INVENTION

The present invention provides methods of treating cancer in a human inneed thereof, comprising determining at least one of the following in asample from said human:

-   -   a. the presence or absence of a mutation at the alanine 677        (A677) residue in EZH2 in a sample from said human; or    -   b. the presence or absence of a mutation at the tyrosine 641        (Y641) residue in EZH2; or    -   c. the presence or absence of an increased level of H3K27me3 as        compared to a control, and        administering to said human an effective amount of an EZH2        inhibitor or a pharmaceutically acceptable salt thereof if at        least one of said A677 mutation, Y641 mutation, or increased        level of H3K27me3 is present in said sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A subset of lymphoma cell lines exhibit grossly elevatedH3K27me3 levels. (A) Global H3K27me3 levels (normalized to total H3)were determined for 111 cancer cell lines from 7 different cancer types.Lymphoma cell lines harboring Y641 mutations are indicated by asterisks.(B) Western blot analysis of global H3K27me3 and H3K27me1 in proteinlysates from a panel of lymphoma cell lines.

FIG. 2. The Pfeiffer lymphoma cell line harbors a heterozygous A677Gmutation in EZH2. (A) Sequence traces from Sanger sequencing of EZH2 inthe Pfeiffer DLBCL cell line and a primary DLBCL patient sample.Heterozygous non-synonymous missense mutation of C₂₀₄₅C/G (asterisks)translates to A677A/G (residue numbering based on NM_001203247). (B)EZH2 domain architecture (Uniprot Q15910). Mutations identified inPfeiffer cells and primary tumors are highlighted. (C) Alignment ofhuman EZH2 with human EZH1, the fly ortholog EZ, and six other relatedSET domain containing histone lysine methyltransferases showing thatY641 and A677 are highly conserved. Dark Grey shading with white lettersrepresents identical residues and boxed amino acids represent conservedresidues. In order for a column to be shaded there has to be 7 out of 9conserved/identical residues in the alignment.

FIG. 3. The A677G EZH2 mutant exhibits activity for all H3K27methylation states. k_(cat) (min⁻¹) for wild type, A677G, and Y641N EZH2using H3K27me0 (black bars), H3K27me1 (gray bars), or H3K27me2 (whitebars) as substrates.

FIG. 4. Exogenous expression of A677G EZH2 stimulates trimethylation ofH3K27. MCF-7 breast cancer cells were transiently transfected withexpression constructs encoding either wild-type or mutant forms of EZH2.(A.) Western blot analysis of H3K27me3, total H3, and EZH2. Actin servesas a loading control. (B.) H3K27me3 levels normalized to histone H3 as apercentage of cells transfected with the empty vector.

FIG. 5. The A677G EZH2 mutation alters the lysine binding pocket througheffects on Y641. A homology model of wild type EZH2 was generated usingthe crystal structure of GLP/EHMT1 bound to an H3K9me2 peptidesubstrate. Modeled structure of the active site region in (A) wild-type,(B) Y641N, and (C) A677G EZH2.

FIG. 6. High levels of H3K27me3 and EZH2 mutation status predictsensitivity to EZH2 inhibition. Lymphoma cell lines were grown in thepresence of the EZH2 inhibitor Compound A for 6 days. The concentrationof Compound A at which a 50% inhibition in growth occurred is indicatedas the growth IC₅₀ and is represented as a line. The H3K27me3 levels arerepresented as a percentage of the H3K27me3 level obtained with the cellline Pfeiffer (bar graph). Cell lines which have a mutation in EZH2either at Y641 or A677 are indicated as black bars while those that arewildtype at these positions are indicated as grey bars.

FIG. 7 Biochemical and cellular mechanistic activity of GSK126. (A)Structure of GSK126. (B) Potency of GSK126 against WT and mutant EZH2.Histone H3 peptides (21-44) with K27me0, K27me1 or K27me2 were used assubstrates (n=2; mean values±s.d. are shown). (C) Effect of GSK126 onH3K27me3 in lymphoma cell lines treated with GSK126 for 48 hours. IC₅₀values were determined using an H3K27me3 ELISA (n≧2; mean values±s.d.are shown). (D) Evaluation of H3K27me3/2/1 in KARPAS-422 followingtreatment for 72 hours. Total histone H3 is shown as a loading control.

FIG. 8. Homology model of EZH2 and predicted binding mode of Compound B(GSK126). (A) A homology model of EZH2 and predicted binding mode ofCompound B (GSK126). GSK126 bound in the SAM binding site is overlaidwith SAH. The H3K27me2 peptide substrate, the SET domain, and thepost-SET domain, and the residue differences between EZH2 and EZH1within 10 Å of the predicted binding mode of GSK126 are indicated. (B) Azoomed in view of the binding mode of GSK126 is depicted. Specifichydrogen bond and arene-H interactions are represented as dashed lines.The binding site surface contributed by residues from the post-SETdomain is shown. (C) A 2D ligand interaction diagram highlightingspecific interactions between residues of EZH2 and GSK126. (D) Diagramof EZH2 functional domains (UniProt Q15910) with the position of theA677 and Y641 activating mutations highlighted within the SET domain.

FIG. 9 Analysis of H3K27 methylation in cell lines treated with GSK126.(A) Comparison of global H3K27me3, H3K27me2, and H3K27me1 levels acrossEZH2 WT (Toledo and SU-DHL-8) and mutant (Pfeiffer and KARPAS-422)lymphoma cell lines. (B) Potency of GSK126 over time as measured byreduction of global H3K27me3 levels in KARPAS-422, Pfeiffer, andSU-DHL-8 B-cell lymphoma cell lines. Cells were treated with a 3-folddilution series of GSK126. The concentration of GSK126 required toreduce H3K27me3 levels by 50% (H3K27me3 IC₅₀) was determined by ELISA(n≧2; mean values±s.d. are shown). (C) Evaluation of H3K27me3, H3K27me2,and H3K27me1 following treatment for 72 hours. Total histone H3 is shownas a loading control.

FIG. 10 Western blot analysis Western blotting of EZH2, SUZ12, and EEDfollowing treatment of EZH2 mutant (a,b) or WT (c,d) lymphoma cell lineswith 0.1% DMSO (vehicle control), 25 nM, 150 nM, 500 nM, or 2 M GSK126for 72 hrs. Actin is included as a loading control.

FIG. 11 GSK126 inhibits the proliferation of several EZH2 mutantlymphoma cell lines. (A) The effect of GSK126 on the growth of 46lymphoma cell lines after 6 days represented as the concentration ofGSK126 required to inhibit 50% of growth (growth IC₅₀). DLBCL, diffuselarge B-cell lymphoma. BL, Burkitt lymphoma. BCBL, AIDS bodycavity-based lymphoma. FL, follicular lymphoma. HL, Hodgkin's lymphoma.NHL, Non-Hodgkin's lymphoma. (B) Potency of GSK126 on growth of Pfeifferand KARPAS-422 over time represented as growth IC₅₀. (C,F)Dose-dependent effects of GSK126 on cell proliferation over time inPfeiffer or KARPAS-422. Growth is expressed as a percentage of CTG attime zero (T₀). (D,G) DNA content histograms showing the effect ofGSK126 on the cell cycle after 72 hours. (E,H) Mean fold-change incaspase 3/7 activity over vehicle control±s.d. is shown (n=4).

FIG. 12. Composite dose-response curves demonstrating the effect ofGSK126 on the growth of 18 DLBCL cell lines. Cell lines were treatedwith varying concentrations of GSK126 for 6 days before cell growth wasevaluated with Cell Titer-Glo (Promega). The y-axis represents thepercent of growth relative to the vehicle control (0.15% DMSO).

FIG. 13 Correlation analysis between inhibition of H3K27me3, cell growthand EZH2 levels (A) Cell growth IC₅₀ values for GSK126 from Table 6plotted against H3K27me3 IC₅₀ values for GSK126 from FIG. 7c . Pearsoncorrelation value is indicated. (B) A representative western blot ofEZH2 and actin from whole cell extracts of lymphoma cell lines. Westernblot signal intensities for EZH2 and actin were quantified using Li-CorOdyssey software. (C) EZH2 signal intensities were normalized for totalactin levels and plotted against cell growth IC₅₀ values for GSK126 in a6 day proliferation assay from Table 6.

FIG. 14 Phenotypic effects of EZH2 knockdown by shRNA. (A) Cellproliferation over a 6 day period of KARPAS-422 (left) and Pfeiffer(right) expressing an shRNA to EZH2 or an on-targeting control shRNA.CTG signal at each timepoint is represented as a percentage of cells atday0 (T0). (B) Caspase3/7 activity over time in KARPAS-422 (left) andPfeiffer (right) expressing an shRNA to EZH2 or a non-targeting controlshRNA. Caspase3/7 activity at each time point is represented as apercentage of activity at day 0 (T0). (C) Western blot analysis of EZH2,H3K27me3, H3K27me2, H3K27me1, and total histone H3 following shRNAknockdown of EZH2. Actin is included as a loading control.

FIG. 15 GSK126 induces transcriptional activation in sensitive celllines. (A) The number of probe sets exhibiting significantly alteredgene expression (FDR<0.1 and fold-change >2 or <−2) following 72 hourtreatment with 500 nM GSK126 (n=2). (B) Basal H3K27me3 ChIP-seqenrichment profiles of genes up-regulated, down-regulated, or all humantranscripts following GSK126 treatment. (C) qRT-PCR analysis of TXNIPand TNFRSF21 following 72 hour treatment with GSK126 (n=3; meanvalues±s.d. are shown). (D) The overlap of up- and down-regulated probesets between 10 DLBCL cell lines using a 2-fold expression changecut-off. (E) Heatmap showing the average gene expression intensities ofthe 35 probe sets exhibiting significantly increased expression in atleast 4 of the 5 most sensitive mutant DLBCL cell lines (Pfeiffer,KARPAS-422, WSU-DLCL2, SU-DHL-10, and SU-DHL-6).

FIG. 16 Expression analysis of DLBCL cell lines. (A) Gene expressionheatmaps of normalized gene expression data for differentially expressedprobe sets following treatment with GSK126 for 72 hours. Darker coloringindicates higher expression. (B) The number of probe sets exhibitingsignificantly altered gene expression (>1.5 fold) following treatment of10 DLBCL cell lines in duplicate for 72 hours with 500 nM GSK126compared with 0.1% DMSO (vehicle control). (C) Correlation between thenumber of up-regulated probe sets and basal H3K27me3 levelsintranscriptionally responsive and unresponsive mutantE ZH2 DLBCLcelllines (Pfeiffer, WSU-DLCL2, KARPAS-422, SU-DHL-10, DB, and SU-DHL-4).H3K27me3 levels are normalized to total histone H3 and are expressed asa percentage of those levels observed in the Pfeiffer cell line.Transcriptionally responsive and unresponsive cell lines are circled.(D) The number of common probe sets within indicated cell linesexhibiting a 1.5 or 2-fold increase in expression with GSK126 treatment.

FIG. 17 Genes up-regulated in response to GSK126 are enriched forH3K27me3. Probe sets that were significantly up-regulated,down-regulated, or unchanged identified in Pfeiffer, WSU-DLCL2, andKARPAS 422 cells following 72 hours with 500 nM GSK126 were mapped toindividual genes and H3K27me3 enrichment determined for each gene and±10 kb from H3K27me3 ChIP-seqdata. Relative H3K27me3 enrichment isrepresented as a white to gray gradient with white representing noenrichment and gray representing the highest enrichment. Each rowrepresents a unique gene.

FIG. 18 Geneontology enrichment analysis. A GO enrichment analysis forprobe sets significantly up-regulated with 500 nM GSK126 in Pfeiffer,WSU-DLCL2, KARPAS-422, SU-DHL-10, or SU-DHL-6. B GO enrichment analysisfor probe sets either significantly up- or downregulated with 500 nMGSK126 in Pfeiffer, WSU-DLCL2, KARPAS-422, SU-DHL-10, or SU-DHL-6 celllines. Over-represented biological process and molecular function termswere filtered for p-value <0.01 (dashedlines), at least 5 genes perterm, and those that were common across ≧3 cell lines.

FIG. 19 In vivo inhibition of H3K27me3 and tumor growth response withGSK126. (A) Response of H3K27me3 in tumor xenografts following 10 daysof QD dosing with GSK126. b qRT-PCR analysis of EZH2 target genes inKARPAS-422 tumor xenografts. Mean values±s.d. (n=3) are shown (A,B). (C)Activity of GSK126 on the growth of subcutaneous KARPAS-422 xenografts.Mean tumor volume±s.e.m. is shown (n=10). (D) Kaplan-Meier survivalcurve of mice treated in (C). Significant P values, calculated using anonparametric log-rank test, between vehicle and treatment groups areindicated. No significant differences were observed between treatmentgroups (p value=0.07-0.32).

FIG. 20 Pharmacokinetic analysis of GSK126. (A) Blood and tumordistribution following intraperitoneal administration of 50 mg/kg GSK126of female beige SCID mice bearing Pfeiffer xenografts. Three mice wereevaluated at each timepoint. (B) Are a under the curve (AUC0-1440),tumor/blood AUC ratio, maximum concentration achieved (Cmax), and timeof maximum concentration (Tmax) for the data presented in a. N/A, notapplicable.

FIG. 21 GSK126 inhibits tumor growth in vivo. (A) Efficacy of GSK126 onthe growth of subcutaneous Pfeiffer xenografts. (B) Efficacy ofintermittent dosing of GSK126 on the growth of subcutaneous KARPAS-422xenografts with or without a 1 week drug holiday. Values are the meantumor volume±standard error (n=10). P values were calculated using anonparametric log-rank test comparing vehicle and each treatment group.

FIG. 22 Effect of GSK126 on bodyweight and peripheral blood. (A-C)Average body weight measurements of mice bearing Pfeiffer (A) orKARPAS-422 (B,C) subcutaneous xenografts during treatment with vehicleor GSK126. Values are represented as a percentage of the average weightat the start of dosing. (D) Complete blood count analysis of CD-1 micefollowing twice weekly dosing over 18 days. RBC, red blood cells (×106cells/μl); HGB, hemoglobin(g/dl); HCT, hematocrit(percent); MCV, meancorpuscle volume (fl); MCH, mean corpuscle hemoglobin (pg); MCHC, meancorpuscle hemoglobin concentration (g/dl); PLT, platelets (×105platelets/μl); WBC, white blood cells (×103 cells/μl); NEUT, neutrophils(×103 cells/μl); LYMPH, lymphocytes (×103 cells/μl); MONO, monocytes(×103 cells/μl); EOS, eosinophils (×103 cells/μl); BASO, basophils (×103cells/μl); LEUK, leukocytes (×103 cells/μl).

FIG. 23 Principal component and correlation analysis of gene expressionprofiling data. (A) PCA plot of data from biological replicates of 10DLBCL cell lines treated for 72 hours with vehicle or 500 nM GSK126. (B)Correlation of biological replicates of DLBCL cell lines with robusttranscriptional changes. K, KARPAS-422; P, Pfeiffer; W, WSU-DLCL2;S10,SU-DHL-10; S6, SU-DHL-6.

DETAILED DESCRIPTION OF THE INVENTION

Recent genome-wide sequencing studies have revealed several genes thatare frequently altered in non-Hodgkin's lymphomas, including EZH2, MLL2,MEF2B, CREBBP, and TP53 among others (1-3). Many of these genes mediate,either directly or indirectly through the recruitment of co-factors, thearray of post-translational modifications observed on the amino-terminaltails of histones. In addition, similar studies have also implicatedthese and other epigenetic factors in transitional cell carcinoma of thebladder (e.g. UTX, ARIDIA, MLL, MLL3), head and neck squamous cellcancers (e.g. EZH2, MLL2), and myeloid malignancies (e.g. IDH1/2, TET2,DNMT3A, EZH2) (4-6). The prevalence of genetic changes affectingtranscription factors and histone modifying genes highlights theimportance of maintaining proper transcriptional regulation intumorigenesis.

The EZH2 gene encodes a SET domain-containing lysine methyltransferasethat along with EED, SUZ12, RbAp48, and AEBP2 forms the PolycombRepressive Complex 2 (PRC2) (7, 8). EZH2 is responsible for methylationof the histone H3 lysine 27 (H3K27) residue which is generallyassociated with transcriptional repression when present in the di- ortri-methylated states (7-9). EZH2 is highly expressed in pro-B cells andprogressively decreases in expression as cells progress into pre-Bcells, immature B cells, and re-circulating B cells (10). EZH2expression is required in the bone marrow for progression of pro-B cellsinto pre-B cells and immature B cells as genetic inactivation of EZH2leads to an accumulation of cells at the pro-B cell stage (10). However,if EZH2 is inactivated after the pro-B cell stage, additional maturationsteps are not hindered suggesting that EZH2 functions early in B-celldifferentiation (10). In fact, multiple groups have shown EZH2 to playan important role in the maintenance of hematopoietic stem andprogenitor cells (11, 12). In particular, EZH2 over-expression inhematopoietic stem cells (HSC) leads to continued self-renewal capacityin serial transplantation models suggesting that EZH2 contributes torepopulating potential and helps cells resist replicative stress (11).

EZH2 is frequently amplified and/or over-expressed in most solid tumortypes (13); however, this does not appear to be the case in lymphomasperhaps owing to the high basal expression of EZH2 in normalproliferating B-cells. Instead EZH2 has been reported to harborrecurrent mutation of the tyrosine 641 (Y641) residue in 22% of germinalcenter B-cell (GCB) diffuse large B-cell lymphoma (DLBCL) and in 7% offollicular lymphomas (FL) (3). Although initially reported to be aloss-of-function mutation (3), subsequent biochemical work demonstrateda novel gain-of-function activity for this Y641 mutant EZH2 (14, 15).While wild-type EZH2 exhibits a strong preference for unmethylated andmono-methylated H3K27 substrates, the Y641 EZH2 mutants observed inlymphomas (Y641F/N/S/H) exhibit profoundly increased activity fordi-methylated substrates and a lack of activity for unmethylated andmono-methylated H3K27 (14, 15). Through the coordinated activities ofwild-type and mutant EZH2 proteins there is a global increase intri-methylation of H3K27 (H3K27me3) in Y641 mutant lymphomas concomitantwith a decrease in mono- and di-methylated H3K27 (14).

These EZH2 Y641 mutations, along with EZH2 over-expression in manytumors, suggest that deregulation of H3K27me3 levels is important inhuman tumorigenesis. Indeed, H3K27me3 levels correlate withprogression-free survival in renal cell carcinoma (16) and diseaseseverity and poor tumor differentiation in esophageal squamous cellcarcinoma (17). In addition to mutation of EZH2 Y641, additionalmechanisms for deregulation of H3K27me3 include inactivating mutationsof the H3K27 demethylase UTX (4, 18, 19) and over-expression of EZH2 dueto multiple mechanisms including decreased miR-101 levels (20, 21),aberrant E2F activity (22), and chromosomal amplification (23).

Through the investigation of global H3K27me3 levels in more than 100cell lines, we have identified a novel EZH2 mutation at the A677 residuethat is responsible for increased H3K27me3 in some lymphoma cells.Characterization of this mutant protein revealed that similar to theY641 EZH2 mutations, exchange of the alanine at position 677 for glycine(A677G) leads to increased activity with a di-methylated H3K27substrate. Importantly, however, this substitution retains criticalinteractions present in wild-type EZH2 leading to efficient utilizationof all H3K27 methylation states including un-, mono-, anddi-methylation. This mutation presents a unique approach for cells toderegulate H3K27 methylation without requiring cooperation withwild-type EZH2 as is the case for Y641 EZH2 mutants.

The present invention provides methods for treating cancer in a human inneed thereof, comprising determining at least one of the following in asample from said human:

-   -   a. the presence or absence of a mutation at the alanine 677        (A677) residue in EZH2 in a sample from said human; or    -   b. the presence or absence of a mutation at the tyrosine 641        (Y641) residue in EZH2; or    -   c. the presence or absence of an increased level of H3K27me3 as        compared to a control,        and administering to said human an effective amount of an EZH2        inhibitor, e.g. a compound of the invention described herein, or        a pharmaceutically acceptable salt thereof if at least one of        said A677 mutation, Y641 mutation, or increased level of        H3K27me3 is present in said sample.

In certain embodiments of the methods herein, at least two of a, b, andc are determined, e.g. a and b, a and c, or b and c, in any order. In afurther embodiment, a, b, and c are each determined, and an EZH2inhibitor, such as a compound of the invention as described herein, isadministered if it is determined that any one of the A677 mutation, theY641 mutation, or an increased level of H3K27me3 as compared to acontrol, is present.

In further embodiments, the presence or absence of a Y641 mutation isdetermined and the Y641 mutation is Y641F, Y641S, Y641H, Y641N, orY641C. In other further embodiments, the presence or absence of an A677mutation is determined and the A677 mutation is A677G.

In further embodiments, an increase in the level of global methylationof a cancer cell or tumor cell is determined. In other embodiments, thelevel of H3K27 methylation are determined. In other embodiments, thelevel of H3K27me0, H3K27me1, H3K27me2, and H3K27me3 are determined andan increase in the level of H3K27me3 suggests treatment with an EZH2inhibitor. In further embodiments of the invention in this paragraph,the levels of methylation are compared to a control, and relativeincrease in methylation relative to a control suggests treatment with anEZH2 inhibitor.

Methods of detecting a mutation in EZH2 at Y641 or A677 are well knownto one of skill in the art and are described herein in the detaileddescription and Examples. Methods of determining an increased level ofmethylation, e.g, H3K27me3, relative to a control are well known in theart and shown in the Examples, and include using an antibody specificfor trimethylated lysine 27 of Histone 3. A control can be any one ofskill in the art would choose, such as a matched cell from a human, amatched tissue from a human, a cell of the same origin as the tumor butknown to have wild type EZH2, or a devised control that correlates withwhat is seen in non-cancerous cells of the same origin or in cells withwild-type EZH2.

In other embodiments of the invention, the sample comprises at least onecancer cell. In certain such embodiments, the sample is a biologicalsample.

In any one of the embodiments of the invention herein, the cancer islymphoma. In further embodiments of the method of the invention, thelymphoma is selected from the group consisting of: germinal centerB-cell (GCB), Diffuse Large B-cell Lymphoma (DLBCL), Splenic marginalzone lymphoma (SMZL), Waldenström's macroglobulinemia lymphoplasmacyticlymphoma (WM), Follicular lymphoma (FL), Mantle Cell Lymphoma (MCL), andExtra nodal marginal zone B-cell lymphoma of mucosa associated lymphoidtissue (MALT).

In embodiments of the methods of the methods of the invention herein,the A677 mutation and/or the Y641 mutation is a somatic mutation.

In other embodiments of the methods of treating cancer, treatmentcomprises an increased response rate and/or an improved progression freesurvival, as compared to an untreated human.

The present invention provides methods of treating cancer in a humanwhich comprises the following steps: (a) detecting the level of H3K27me3from at least one tumor cell from said human and (b) administering tosaid human an effective amount of an EZH2 inhibitor or apharmaceutically acceptable salt thereof in a pharmaceutical compositionif said at least one tumor cell has a high level of H3K27me3.

The present invention also relates to a method of treating cancer in ahuman which comprises the following steps: (a) performing a genotypingtechnique on a biological sample from the subject tumor to determinewhether said tumor has somatic mutations of EZH2 at the tyrosine 641(Y641) residue; and (b) correlating the detection of said mutations withincreased likelihood of increased response rate and/or prolongedprogression free survival when administered an EZH2 inhibitor.

The present invention also relates to a method of treating cancer in ahuman which comprises the following steps: (a) performing a genotypingtechnique on a biological sample from the subject tumor to determinewhether said tumor has somatic mutations of EZH2 at the tyrosine 641(Y641) residue; and (b) administer an effective amount of an EZH2inhibitor or a pharmaceutically acceptable salt thereof to said human ifsaid tumor has a mutation of EZH2 at the tyrosine 641 residue.

The present invention also relates to a method of treating cancer in ahuman which comprises the following steps: (a) performing a genotypingtechnique on a biological sample from the subject tumor to determinewhether said tumor has somatic mutations of EZH2 at the tyrosine 641(Y641) residue; and (b) administer an effective amount of an EZH2inhibitor or a pharmaceutically acceptable salt thereof to said human ifsaid tumor has a mutation of EZH2 at the tyrosine 641 residue, whereinsuch EZH2 mutation is Y641N, Y641F, Y641 S, Y641H, or Y641C.

The present invention also relates to a method of treating cancer in ahuman which comprises the following steps: (a) performing a genotypingtechnique on a biological sample from the subject tumor to determinewhether said tumor has somatic mutations of EZH2 at the A677 residue;and (b) correlating the detection of said mutations with increasedlikelihood of increased response rate and/or prolonged progression freesurvival when administered an EZH2 inhibitor.

The present invention also relates to a method of treating cancer in ahuman which comprises the following steps: (a) performing a genotypingtechnique on a biological sample from the subject tumor to determinewhether said tumor has somatic mutations of EZH2 at the A677 residue;and (b) administering an effective amount of an EZH2 inhibitor or apharmaceutically acceptable salt thereof to said human if said tumor hasa mutation of EZH2 at the A677 residue.

The present invention also relates to a method of treating cancer in ahuman which comprises the following steps: (a) performing a genotypingtechnique on a biological sample from the subject tumor to determinewhether said tumor has somatic mutations of EZH2 at the A677 residue;and (b) administering an effective amount of an EZH2 inhibitor or apharmaceutically acceptable salt thereof to said human if said tumor hasa mutation of EZH2 at the A677 residue; wherein such EZH2 mutation isA677G.

Definitions

The term “wild type” as is understood in the art refers to a polypeptideor polynucleotide sequence that occurs in a native population withoutgenetic modification. As is also understood in the art, a “variant”includes a polypeptide or polynucleotide sequence having at least onemodification to an amino acid or nucleic acid compared to thecorresponding amino acid or nucleic acid found in a wild typepolypeptide or polynucleotide, respectively. Included in the termvariant is Single Nucleotide Polymorphism (SNP) where a single base pairdistinction exists in the sequence of a nucleic acid strand compared tothe most prevalently found (wild type) nucleic acid strand.

As used herein “genetic modification” or “genetically modified” orgrammatical variations thereof refers to, but is not limited to, anysuppression, substitution, amplification, deletion and/or insertion ofone or more bases into DNA sequence(s). Also, as used herein“genetically modified” can refer to a gene encoding a polypeptide or apolypeptide having at least one deletion, substitution or suppression ofa nucleic acid or amino acid, respectively. Genetic variants and/or SNPscan be identified by known methods. For example, wild type or SNPs canbe identified by DNA amplification and sequencing techniques, DNA andRNA detection techniques, including, but not limited to Northern andSouthern blot, respectively, and/or various biochip and arraytechnologies. WT and mutant polypeptides can be detected by a variety oftechniques including, but not limited to immunodiagnostic techniquessuch as ELISA and western Blot. As used herein, the process of detectingan allele or polymorphism includes but is not limited to serologic andgenetic methods. The allele or polymorphism detected may be functionallyinvolved in affecting an individual's phenotype, or it may be an alleleor polymorphism that is in linkage disequilibrium with a functionalpolymorphism/allele. Polymorphisms/alleles are evidenced in the genomicDNA of a subject, but may also be detectable from RNA, cDNA or proteinsequences transcribed or translated from this region, as will beapparent to one skilled in the art.

As is well known genetics, nucleotide and related amino acid sequencesobtained from different sources for the same gene may vary both in thenumbering scheme and in the precise sequence. Such differences may bedue to numbering schemes, inherent sequence variability within the gene,and/or to sequencing errors. Accordingly, reference herein to aparticular polymorphic site by number will be understood by those ofskill in the art to include those polymorphic sites that correspond insequence and location within the gene, even where differentnumbering/nomenclature schemes are used to describe them.

As used herein, “genotyping” a subject (or DNA or other sample) for apolymorphic allele of a gene(s) or a mutation in at least onepolypeptide or gene encoding at least one polypeptide means detectingwhich mutated, allelic or polymorphic form(s) of the gene(s) or geneexpression products (e.g., hnRNA, mRNA or protein) are present or absentin a subject (or a sample). Related RNA or protein expressed from suchgene may also be used to detect mutant or polymorphic variation. As iswell known in the art, an individual may be heterozygous or homozygousfor a particular allele. More than two allelic forms may exist, thusthere may be more than three possible genotypes. As used herein, anallele may be ‘detected’ when other possible allelic variants have beenruled out; e.g., where a specified nucleic acid position is found to beneither adenine (A), thymine (T) or cytosine (C), it can be concludedthat guanine (G) is present at that position (i.e., G is ‘detected’ or‘diagnosed’ in a subject). Sequence variations may be detected directly(by, e.g., sequencing) or indirectly (e.g., by restriction fragmentlength polymorphism analysis, or detection of the hybridization of aprobe of known sequence, or reference strand conformation polymorphism),or by using other known methods.

As used herein, a “genetic subset” of a population consists of thosemembers of the population having a particular genotype or a tumor havingat least one somatic mutation. In the case of a biallelic polymorphism,a population can potentially be divided into three subsets: homozygousfor allele 1 (1,1), heterozygous (1,2), and homozygous for allele 2(2,2). A ‘population’ of subjects may be defined using various criteria.

As used herein, a human that is in need of treatment for cancer, may be“predisposed to” or “at increased risk of” a particular phenotypicresponse based on genotyping will be more likely to display thatphenotype than an individual with a different genotype at the targetpolymorphic locus (or loci). Where the phenotypic response is based on amulti-allelic polymorphism, or on the genotyping of more than one gene,the relative risk may differ among the multiple possible genotypes.

A human that is in need of treatment for cancer may alternatively have atumor or cancer cells with somatic mutations, and genotyping or otherdetection of the mutations can be performs.

As used herein “response” to treatment and grammatical variationsthereof, includes but is not limited to an improved clinical conditionof a patient after the patient received medication. Response can alsomean that a patient's condition does not worsen upon that start oftreatment. Response can be defined by the measurement of certainmanifestations of a disease or disorder. With respect to cancer,response can mean, but is not limited to, a reduction of the size and ornumber of tumors and/or tumor cells in a patient. Response can also bedefined by a other endpoints such as a reduction or attenuation in thenumber of pre-tumorous cells in a patient.

“Genetic testing” (also called genetic screening) as used herein refersto the testing of a biological sample from a subject to determine thesubject's genotype; and may be utilized to determine if the subject'sgenotype comprises alleles that either cause, or increase susceptibilityto, a particular phenotype (or that are in linkage disequilibrium withallele(s) causing or increasing susceptibility to that phenotype).

Samples, e.g. biological samples, for testing or determining of one ormore mutations may be selected from the group of proteins, nucleotides,cellular blebs or components, serum, cells, blood, blood components,urine and saliva. Testing for mutations may be conducted by severaltechniques known in the art and/or described herein.

The sequence of any nucleic acid including a gene or PCR product or afragment or portion thereof may be sequenced by any method known in theart (e.g., chemical sequencing or enzymatic sequencing). “Chemicalsequencing” of DNA may denote methods such as that of Maxam and Gilbert(1977) (Proc. Natl. Acad. Sci. USA 74:560), in which DNA is randomlycleaved using individual base-specific reactions. “Enzymatic sequencing”of DNA may denote methods such as that of Sanger (Sanger, et al., (1977)Proc. Natl. Acad. Sci. USA 74:5463).

Conventional molecular biology, microbiology, and recombinant DNAtechniques including sequencing techniques are well known among thoseskilled in the art. Such techniques are explained fully in theliterature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning:A Laboratory Manual, Second Edition (1989) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (herein “Sambrook, et al., 1989”); DNACloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985);Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. (1985)); TranscriptionAnd Translation (B. D. Hames & S. J. Higgins, eds. (1984)); Animal CellCulture (R. I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRLPress, (1986)); B. Perbal, A Practical Guide To Molecular Cloning(1984); F. M. Ausubel, et al. (eds.), Current Protocols in MolecularBiology, John Wiley & Sons, Inc. (1994

The Peptide Nucleic Acid (PNA) affinity assay is a derivative oftraditional hybridization assays (Nielsen et al., Science 254:1497-1500(1991); Egholm et al., J. Am. Chem. Soc. 114:1895-1897 (1992); James etal., Protein Science 3:1347-1350 (1994)). PNAs are structural DNA mimicsthat follow Watson-Crick base pairing rules, and are used in standardDNA hybridization assays. PNAs display greater specificity inhybridization assays because a PNA/DNA mismatch is more destabilizingthan a DNA/DNA mismatch and complementary PNA/DNA strands form strongerbonds than complementary DNA/DNA strands.

DNA microarrays have been developed to detect genetic variations andpolymorphisms (Taton et al., Science 289:1757-60, 2000; Lockhart et al.,Nature 405:827-836 (2000); Gerhold et al., Trends in BiochemicalSciences 24:168-73 (1999); Wallace, R. W., Molecular Medicine Today3:384-89 (1997); Blanchard and Hood, Nature Biotechnology 149:1649(1996)). DNA microarrays are fabricated by high-speed robotics, on glassor nylon substrates, and contain DNA fragments with known identities(“the probe”). The microarrays are used for matching known and unknownDNA fragments (“the target”) based on traditional base-pairing rules.

The terms “polypeptide” and “protein” are used interchangeably and areused herein as a generic term to refer to native protein, fragments,peptides, or analogs of a polypeptide sequence. Hence, native protein,fragments, and analogs are species of the polypeptide genus.

The terminology “X#Y” in the context of a mutation in a polypeptidesequence is art-recognized, where “#” indicates the location of themutation in terms of the amino acid number of the polypeptide, “X”indicates the amino acid found at that position in the wild-type aminoacid sequence, and “Y” indicates the mutant amino acid at that position.For example, the notation “G12S” with reference to the K-ras polypeptideindicates that there is a glycine at amino acid number 12 of thewild-type K-ras sequence, and that glycine is replaced with a serine inthe mutant K-ras sequence.

A “mutation” in a polypeptide or a gene encoding a polypeptide andgrammatical variations thereof means a polypeptide or gene encoding apolypeptide having one or more allelic variants, splice variants,derivative variants, substitution variants, deletion variants, and/orinsertion variants, fusion polypeptides, orthologs, and/or interspecieshomologs. By way of example, at least one mutation of EZH2 would includean EZH2 in which part of all of the sequence of a polypeptide or geneencoding the polypeptide is absent or not expressed in the cell for atleast one of the EZH2 proteins produced in the cell. For example, anEZH2 protein may be produced by a cell in a truncated form and thesequence of the truncated form may be wild type over the sequence of thetruncate. A deletion may mean the absence of all or part of a gene orprotein encoded by a gene. An EZH2 mutation also means a mutation at asingle base in a polynucleotide, or a single amino acid substitution.Additionally, some of a protein expressed in or encoded by a cell may bemutated, e.g., at a single amino acid, while other copies of the sameprotein produced in the same cell may be wild type.

Mutations may be detected in the polynucleotide or translated protein bya number of methods well known in the art. These methods include, butare not limited to, sequencing, RT-PCR, and in situ hybridization, suchas fluorescence-based in situ hybridization (FISH), antibody detection,protein degradation sequencing, etc. Epigenetic changes, such asmethylation states, may also result in mutations and/or lack ofexpression of part or all of a protein from the correspondingpolynucleotide encoding it.

As used herein “genetic abnormality” is meant a deletion, substitution,addition, translocation, amplification and the like relative to thenormal native nucleic acid content of a cell of a subject. As usedherein “gene encoding an EZH2 protein” means any part of a gene orpolynucleotide encoding any EZH2 protein. Included within the meaning ofthis term are exons encoding EZH2. Gene encoding EZH2 proteins includebut are not limited to genes encoding part or all of EZH2.

The term “polynucleotide” as referred to herein means a polymeric formof nucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms of DNA.

The term “oligonucleotide” referred to herein includes naturallyoccurring and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. Preferably oligonucleotides are 10 to 60bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or20 to 40 bases in length. Oligonucleotides are usually single stranded,e.g. for probes, although oligonucleotides may be double stranded, e.g.for use in the construction of a gene mutant. Oligonucleotides can beeither sense or antisense oligonucleotides.

An oligonucleotide probe, or probe, is a nucleic acid molecule whichtypically ranges in size from about 8 nucleotides to several hundrednucleotides in length. Such a molecule is typically used to identify atarget nucleic acid sequence in a sample by hybridizing to such targetnucleic acid sequence under stringent hybridization conditions.Hybridization conditions have been described in detail above.

PCR primers are also nucleic acid sequences, although PCR primers aretypically oligonucleotides of fairly short length which are used inpolymerase chain reactions. PCR primers and hybridization probes canreadily be developed and produced by those of skill in the art, usingsequence information from the target sequence. (See, for example,Sambrook et al., supra or Glick et al., supra).

As used herein “overexpressed” and “overexpression” and grammaticalvariations thereof means that a given cell produces an increased numberof a certain protein relative to a normal cell. For instance, some tumorcells are known to overexpress Her2 or Erb2 on the cell surface comparedwith cells from normal breast tissue. Gene transfer experiments haveshown that overexpression of HER2 will transform NIH 3T3 cells and alsocause an increase in resistance to the toxic macrophage cytokine tumornecrosis factor. Hudziak et al., “Amplified Expression of the HER2/ERBB2Oncogene Induces Resistance to Tumor Necrosis Factor Alpha in NIH 3T3Cells”, Proc. Natl. Acad. Sci. USA 85, 5102-5106 (1988). Expressionlevels of a polypeptide in a particular cell can be effected by, but notlimited to, mutations, deletions and/or substitutions of variousregulatory elements and/or non-encoding sequence in the cell genome.

As used herein, “treatment” means any manner in which one or moresymptoms associated with the disorder are beneficially altered.Accordingly, the term includes healing or amelioration of a symptom orside effect of the disorder or a decrease in the rate of advancement ofthe disorder.

As used herein, the terms “cancer,” “neoplasm,” and “tumor,” are usedinterchangeably and in either the singular or plural form, refer tocells that have undergone a malignant transformation that makes thempathological to the host organism. Primary cancer cells (that is, cellsobtained from near the site of malignant transformation) can be readilydistinguished from non-cancerous cells by well-established techniques,particularly histological examination. The definition of a cancer cell,as used herein, includes not only a primary cancer cell, but any cellderived from a cancer cell ancestor. This includes metastasized cancercells, and in vitro cultures and cell lines derived from cancer cells.When referring to a type of cancer that normally manifests as a solidtumor, a “clinically detectable” tumor is one that is detectable on thebasis of tumor mass; e.g., by procedures such as CAT scan, MR imaging,X-ray, ultrasound or palpation, and/or which is detectable because ofthe expression of one or more cancer-specific antigens in a sampleobtainable from a patient. Tumors may be hematopoietic tumor, forexample, tumors of blood cells or the like. Specific examples ofclinical conditions based on such a tumor include leukemia such aschronic myelocytic leukemia or acute myelocytic leukemia; myeloma suchas multiple myeloma; lymphoma and the like.

The cancer may be any cancer in which an abnormal number of blast cellsare present or that is diagnosed as a haematological cancer ordysplasia, such as leukemia, myeloid malignancy or myeloid dysplasia,including but not limited to, undifferentiated acute myelogenousleukemia, myeloblastic leukemia, myeloblastic leukemia, promyelocyticleukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemiaand megakaryoblastic leukemia. In one aspect, the cancer is a myeloidmalignancy cancer. In another aspect, the cancer is leukemia. Theleukemia may be acute lymphocytic leukemia, acute non-lymphocyticleukemia, acute myeloid leukemia (AML), chronic lymphocytic leukemia,chronic myelogenous (or myeloid) leukemia (CML), and chronicmyelomonocytic leukemia (CMML). In one embodiment, the human hasagnogenic myeloid metaplasia and/or poor-risk myelodysplasia (MDS). Insome aspects the cancer is relapsed or refractory.

Hematopoietic cancers also include lymphoid malignancies, which mayaffect the lymph nodes, spleens, bone marrow, peripheral blood, and/orextranodal sites. Lymphoid cancers include B-cell malignancies, whichinclude, but are not limited to, B-cell non-Hodgkin's lymphomas(B-NHLs). B-NHLs may be indolent (or low-grade), intermediate-grade (oraggressive) or high-grade (very aggressive). Indolent B cell lymphomasinclude follicular lymphoma (FL); small lymphocytic lymphoma (SLL);marginal zone lymphoma (MZL) including nodal MZL, extranodal MZL,splenic MZL and splenic MZL with villous lymphocytes; lymphoplasmacyticlymphoma (LPL); and mucosa-associated-lymphoid tissue (MALT orextranodal marginal zone) lymphoma. Intermediate-grade B-NHLs includemantle cell lymphoma (MCL) with or without leukemic involvement, diffuselarge cell lymphoma (DLBCL), follicular large cell (or grade 3 or grade3B) lymphoma, and primary mediastinal lymphoma (PML). High-grade B-NHLsinclude Burkitt's lymphoma (BL), Burkitt-like lymphoma, smallnon-cleaved cell lymphoma (SNCCL) and lymphoblastic lymphoma. OtherB-NHLs include immunoblastic lymphoma (or immunocytoma), primaryeffusion lymphoma, HIV associated (or AIDS related) lymphomas, andpost-transplant lymphoproliferative disorder (PTLD) or lymphoma. B-cellmalignancies also include, but are not limited to, chronic lymphocyticleukemia (CLL), prolymphocytic leukemia (PLL), Waldenström'smacroglobulinemia (WM), hairy cell leukemia (HCL), large granularlymphocyte (LGL) leukemia, acute lymphoid (or lymphocytic orlymphoblastic) leukemia, and Castleman's disease. NHL may also includeT-cell non-Hodgkin's lymphoma s(T-NHLs), which include, but are notlimited to T-cell non-Hodgkin's lymphoma not otherwise specified (NOS),peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma(ALCL), angioimmunoblastic lymphoid disorder (AILD), nasal naturalkiller (NK) cell/T-cell lymphoma, gamma/delta lymphoma, cutaneous T celllymphoma, mycosis fungoides, and Sezary syndrome.

Hematopoietic cancers also include Hodgkin's lymphoma (or disease)including classical Hodgkin's lymphoma, nodular sclerosing Hodgkin'slymphoma, mixed cellularity Hodgkin's lymphoma, lymphocyte predominant(LP) Hodgkin's lymphoma, nodular LP Hodgkin's lymphoma, and lymphocytedepleted Hodgkin's lymphoma. Hematopoietic cancers also include plasmacell diseases or cancers such as multiple myeloma (MM) includingsmoldering MM, monoclonal gammopathy of undetermined (or unknown orunclear) significance (MGUS), plasmacytoma (bone, extramedullary),lymphoplasmacytic lymphoma (LPL), Waldenström's Macroglobulinemia,plasma cell leukemia, and primary amyloidosis (AL). Hematopoieticcancers may also include other cancers of additional hematopoieticcells, including polymorphonuclear leukocytes (or neutrophils),basophils, eosinophils, dendritic cells, platelets, erythrocytes andnatural killer cells. Tissues which include hematopoietic cells referredherein to as “hematopoietic cell tissues” include bone marrow;peripheral blood; thymus; and peripheral lymphoid tissues, such asspleen, lymph nodes, lymphoid tissues associated with mucosa (such asthe gut-associated lymphoid tissues), tonsils, Peyer's patches andappendix, and lymphoid tissues associated with other mucosa, forexample, the bronchial linings.

In some embodiments, the sample is selected from the group consisting ofcancer cells, tumor cells, cells, blood, blood components, urine andsaliva.

Compounds of the Invention

In certain embodiments of the methods of treating cancer in a human inneed thereof, the EZH2 inhibitor is of Formula I:

wherein:

W is N or CR²;

X and Z are each independently selected from the group consisting ofhydrogen, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, unsubstituted orsubstituted (C₃-C₈)cycloalkyl, unsubstituted or substituted(C₃-C₈)cycloalkyl-(C₁-C₈)alkyl or —(C₂-C₈)alkenyl, unsubstituted orsubstituted (C₅-C₈)cycloalkenyl, unsubstituted or substituted(C₅-C₈)cycloalkenyl-(C₁-C₈)alkyl or —(C₂-C₈)alkenyl,(C₆-C₁₀)bicycloalkyl, unsubstituted or substituted heterocycloalkyl,unsubstituted or substituted heterocycloalkyl-(C₁-C₈)alkyl or—(C₂-C₈)alkenyl, unsubstituted or substituted aryl, unsubstituted orsubstituted aryl-(C₁-C₈)alkyl or —(C₂-C₈)alkenyl, unsubstituted orsubstituted heteroaryl, unsubstituted or substitutedheteroaryl-(C₁-C₈)alkyl or —(C₂-C₈)alkenyl, halogen, cyano, —COR^(a),—CO₂R^(a), —CONR^(a)R^(b), —CONR^(a)NR^(a)R^(b), —SR^(a), —SOR^(a),—SO₂R^(a), —SO₂NR^(a)R^(b), nitro, —NR^(a)R^(b), —NR^(a)C(O)R^(b),—NR^(a)C(O)NR^(a)R^(b), —NR^(a)C(O)OR^(a), —NR^(a)SO₂R^(b),—NR^(a)SO₂NR^(a)R^(b), —NR^(a)NR^(a)R^(b), —NR^(a)NR^(a)C(O)R^(b),—NR^(a)NR^(a)C(O)NR^(a)R^(b), —NR^(a)NR^(a)C(O)OR^(a), —OR^(a),—OC(O)R^(a), and —OC(O)NR^(a)R^(b);

Y is hydrogen or halogen;

R¹ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, unsubstituted orsubstituted (C₃-C₈)cycloalkyl, unsubstituted or substituted(C₃-C₈)cycloalkyl-(C₁-C₈)alkyl or —(C₂-C₈)alkenyl, unsubstituted orsubstituted (C₅-C₈)cycloalkenyl, unsubstituted or substituted(C₅-C₈)cycloalkenyl-(C₁-C₈)alkyl or —(C₂-C₈)alkenyl, unsubstituted orsubstituted (C₆-C₁₀)bicycloalkyl, unsubstituted or substitutedheterocycloalkyl or —(C₂-C₈)alkenyl, unsubstituted or substitutedheterocycloalkyl-(C₁-C₈)alkyl, unsubstituted or substituted aryl,unsubstituted or substituted aryl-(C₁-C₈)alkyl or —(C₂-C₈)alkenyl,unsubstituted or substituted heteroaryl, unsubstituted or substitutedheteroaryl-(C₁-C₈)alkyl or —(C₂-C₈)alkenyl, —COR^(a), —CO₂R^(a),—CONR^(a)R^(b), or —CONR^(a)NR^(a)R^(b);

When present R² is hydrogen, (C₁-C₈)alkyl, trifluoromethyl, alkoxy, orhalogen, in which said (C₁-C₈)alkyl may be substituted with one to twogroups selected from amino and (C₁-C₃)alkylamino;

R⁷ is hydrogen, (C₁-C₃)alkyl, or alkoxy; R³ is hydrogen, (C₁-C₈)alkyl,cyano, trifluoromethyl, —NR^(a)R^(b), or halogen;

R⁶ is selected from the group consisting of hydrogen, halo,(C₁-C₈)alkyl, (C₂-C₈)alkenyl, —B(OH)₂, substituted or unsubstituted(C₂-C₈)alkynyl, unsubstituted or substituted (C₃-C₈)cycloalkyl,unsubstituted or substituted (C₃-C₈)cycloalkyl-(C₁-C₈)alkyl,unsubstituted or substituted (C₅-C₈)cycloalkenyl, unsubstituted orsubstituted (C₅-C₈)cycloalkenyl-(C₁-C₈)alkyl, (C₆-C₁₀)bicycloalkyl,unsubstituted or substituted heterocycloalkyl, unsubstituted orsubstituted heterocycloalkyl-(C₁-C₈)alkyl, unsubstituted or substitutedaryl, unsubstituted or substituted aryl-(C₁-C₈)alkyl, unsubstituted orsubstituted heteroaryl, unsubstituted or substitutedheteroaryl-(C₁-C₈)alkyl, cyano, —COR^(a), —CO₂R^(a), —CONR^(a)R^(b),—CONR^(a)NR^(a)R^(b), —SR^(a), —SOR^(a), —SO₂R^(a), —SO₂NR^(a)R^(b),nitro, —NR^(a)R^(b), —NR^(a)C(O)R^(b), —NR^(a)C(O)NR^(a)R^(b),—NR^(a)C(O)OR^(a), —NR^(a)SO₂R^(b), —NR^(a)SO₂NR^(a)R^(b),—NR^(a)NR^(a)R^(b), —NR^(a)NR^(a)C(O)R^(b),—NR^(a)NR^(a)C(O)NR^(a)R^(b), —NR^(a)NR^(a)C(O)OR^(a), —OR^(a),—OC(O)R^(a), and —OC(O)NR^(a)R^(b);

-   -   wherein any (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,        cycloalkyl, cycloalkenyl, bicycloalkyl, heterocycloalkyl, aryl,        or heteroaryl group is optionally substituted by 1, 2 or 3        groups independently selected from the group consisting of        —O(C₁-C₆)alkyl(R^(c))₁₋₂, —S(C₁-C₆)alkyl(R^(c))₁₋₂,        —(C₁-C₆)alkyl(R^(c))₁₋₂, (C₁-C₈)alkyl-heterocycloalkyl,        (C₃-C₈)cycloalkyl-heterocycloalkyl, halogen, (C₁-C₆)alkyl,        (C₃-C₈)cycloalkyl, (C₅-C₈)cycloalkenyl, (C₁-C₆)haloalkyl, cyano,        —COR^(a), —CO₂R^(a), —CONR^(a)R^(b), —SR^(a), —SOR^(a),        —SO₂R^(a), —SO₂NR^(a)R^(b), nitro, —NR^(a)R^(b),        —NR^(a)C(O)R^(b), —NR^(a)C(O)NR^(a)R^(b), —NR^(a)C(O)OR^(a),        —NR^(a)SO₂R^(b), —NR^(a)SO₂NR^(a)R^(b), —OR^(a), —OC(O)R^(a),        —OC(O)NR^(a)R^(b), heterocycloalkyl, aryl, heteroaryl,        aryl(C₁-C₄)alkyl, and heteroaryl(C₁-C₄)alkyl;        -   wherein any aryl or heteroaryl moiety of said aryl,            heteroaryl, aryl(C₁-C₄)alkyl, or heteroaryl(C₁-C₄)alkyl is            optionally substituted by 1, 2 or 3 groups independently            selected from the group consisting of halogen, (C₁-C₆)alkyl,            (C₃-C₈)cycloalkyl, (C₅-C₈)cycloalkenyl, (C₁-C₆)haloalkyl,            cyano, —COR^(a), —CO₂R^(a), —CONR^(a)R^(b), —SR^(a),            —SOR^(a), —SO₂R^(a), —SO₂NR^(a)R^(b), nitro, —NR^(a)R^(b),            —NR^(a)C(O)R^(b), —NR^(a)C(O)NR^(a)R^(b), —NR^(a)C(O)OR^(a),            —NR^(a)SO₂R^(b), —NR^(a)SO₂NR^(a)R^(b), —OR^(a),            —OC(O)R^(a), and —OC(O)NR^(a)R^(b);

each R^(c) is independently (C₁-C₄)alkylamino, —NR^(a)SO₂R^(b),—SOR^(a), —SO₂R^(a), —NR^(a)C(O)OR^(a), —NR^(a)R^(b), or —CO₂R^(a);

R^(a) and R^(b) are each independently hydrogen, (C₁-C₈)alkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)cycloalkyl, (C₅-C₈)cycloalkenyl,(C₆-C₁₀)bicycloalkyl, heterocycloalkyl, aryl, heteroaryl, wherein said(C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, cycloalkyl, cycloalkenyl,bicycloalkyl, heterocycloalkyl, aryl, or heteroaryl group is optionallysubstituted by 1, 2 or 3 groups independently selected from halogen,hydroxyl, (C₁-C₄)alkoxy, amino, (C₁-C₄)alkylamino,((C₁-C₄)alkyl)((C₁-C₄)alkyl)amino, —CO₂H, —CO₂(C₁-C₄)alkyl, —CONH₂,—CONH(C₁-C₄)alkyl, —CON((C₁-C₄)alkyl)((C₁-C₄)alkyl), —SO₂(C₁-C₄)alkyl,—SO₂NH₂, —SO₂NH(C₁-C₄)alkyl, or —SO₂N((C₁-C₄)alkyl)((C₁-C₄)alkyl);

or R^(a) and R^(b) taken together with the nitrogen to which they areattached represent a 5-8 membered saturated or unsaturated ring,optionally containing an additional heteroatom selected from oxygen,nitrogen, and sulfur, wherein said ring is optionally substituted by 1,2, or 3 groups independently selected from (C₁-C₄)alkyl,(C₁-C₄)haloalkyl, amino, (C₁-C₄)alkylamino,((C₁-C₄)alkyl)((C₁-C₄)alkyl)amino, hydroxyl, oxo, (C₁-C₄)alkoxy, and(C₁-C₄)alkoxy(C₁-C₄)alkyl, wherein said ring is optionally fused to a(C₃-C₈)cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring;

or R^(a) and R^(b) taken together with the nitrogen to which they areattached represent a 6- to 10-membered bridged bicyclic ring systemoptionally fused to a (C₃-C₈)cycloalkyl, heterocycloalkyl, aryl, orheteroaryl ring;

or a pharmaceutically acceptable salt thereof.

Compounds of Formula I, and methods of making the same are disclosed inWO2011/140324, which is incorporated by reference in its entiretyherein.

In a further embodiment, EZH2 inhibitor is a compound of Formula (I)wherein W is CR², or a pharmaceutically acceptable salt thereof.

In yet a further embodiment, the EZH2 inhibitor is a Compound of FormulaI having Formula B:

or a pharmaceutically acceptable salt thereof.

Compounds having Formula B and methods of making the same are disclosedin WO 2011/140324, e.g. Example 270.

In another embodiment, the EZH2 inhibitor is Compound A having formula1-(1-methylethyl)-N-[(6-methyl-2-oxo-4-propyl-1,2-dihydro-3-pyridinyl)methyl]-6-[6-(4-methyl-1-piperazinyl)-3-pyridinyl]-1H-indazole-4-carboxamide;

Additional EZH2 inhibitors are well known in the art. For example, EZH2inhibitors are disclosed in WO 2011/140324, WO 2011/140325 and WO2012/075080, each of which is incorporated by reference herein in itsentirety. In any of the embodiments herein, the EZH2 inhibitor may be acompound disclosed in WO 2011/140324, WO 2011/140325 or WO 2012/075080.

For the avoidance of doubt, unless otherwise indicated, the term“substituted” means substituted by one or more defined groups. In thecase where groups may be selected from a number of alternative groupsthe selected groups may be the same or different.

The term “independently” means that where more than one substituent isselected from a number of possible substituents, those substituents maybe the same or different.

An “effective amount” means that amount of a drug or pharmaceuticalagent that will elicit the biological or medical response of a tissue,system, animal or human that is being sought, for instance, by aresearcher or clinician. Furthermore, the term “therapeuticallyeffective amount” means any amount which, as compared to a correspondingsubject who has not received such amount, results in improved treatment,healing, prevention, or amelioration of a disease, disorder, or sideeffect, or a decrease in the rate of advancement of a disease ordisorder. The term also includes within its scope amounts effective toenhance normal physiological function.

As used herein the term “alkyl” refers to a straight- or branched-chainhydrocarbon radical having the specified number of carbon atoms, so forexample, as used herein, the terms “C₁C₈alkyl” refers to an alkyl grouphaving at least 1 and up to 8 carbon atoms respectively. Examples ofsuch branched or straight-chained alkyl groups useful in the presentinvention include, but are not limited to, methyl, ethyl, n-propyl,isopropyl, isobutyl, n-butyl, t-butyl, n-pentyl, isopentyl, n-hexyl,n-heptyl, and n-octyl and branched analogs of the latter 5 normalalkanes.

The term “alkoxy” as used herein means —O(C₁C₈alkyl) including —OCH₃,—OCH₂CH₃ and —OC(CH₃)₃ and the like per the definition of alkyl above.

The term “alkylthio” as used herein is meant —S(C₁C₈alkyl) including—SCH₃, —SCH₂CH₃ and the like per the definition of alkyl above.

The term “acyloxy” means —OC(O)C₁C₈alkyl and the like per the definitionof alkyl above.

“Acylamino” means-N(H)C(O)C₁C₈alkyl and the like per the definition ofalkyl above.

“Aryloxy” means —O(aryl), —O(substituted aryl), —O(heteroaryl) or—O(substituted heteroaryl).

“Arylamino” means —NH(aryl), —NH(substituted aryl), —NH(heteroaryl) or—NH(substituted heteroaryl), and the like.

When the term “alkenyl” (or “alkenylene”) is used it refers to straightor branched hydrocarbon chains containing the specified number of carbonatoms and at least 1 and up to 5 carbon-carbon double bonds. Examplesinclude ethenyl (or ethenylene) and propenyl (or propenylene).

When the term “alkynyl” (or “alkynylene”) is used it refers to straightor branched hydrocarbon chains containing the specified number of carbonatoms and at least 1 and up to 5 carbon-carbon triple bonds. Examplesinclude ethynyl (or ethynylene) and propynyl (or propynylene).

“Haloalkyl” refers to an alkyl group group that is substituted with oneor more halogen substituents, suitably from 1 to 6 substituents.Haloalkyl includes trifluoromethyl.

When “cycloalkyl” is used it refers to a non-aromatic, saturated, cyclichydrocarbon ring containing the specified number of carbon atoms. So,for example, the term “C₃-C₈cycloalkyl” refers to a non-aromatic cyclichydrocarbon ring having from three to eight carbon atoms. Exemplary“C₃-C₈cycloalkyl” groups useful in the present invention include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl and cyclooctyl.

The term “C₅C₈cycloalkenyl” refers to a non-aromatic monocycliccarboxycyclic ring having the specified number of carbon atoms and up to3 carbon-carbon double bonds. “Cycloalkenyl” includes by way of examplecyclopentenyl and cyclohexenyl.

Where “C₃C₈heterocycloalkyl” is used, it means a non-aromaticheterocyclic ring containing the specified number of ring atoms being,saturated or having one or more degrees of unsaturation and containingone or more heteroatom substitutions independently selected from O, Sand N. Such a ring may be optionally fused to one or more other“heterocyclic” ring(s) or cycloalkyl ring(s). Examples are given hereinbelow.

“Aryl” refers to optionally substituted monocyclic or polycarbocyclicunfused or fused groups having 6 to 14 carbon atoms and having at leastone aromatic ring that complies with Hückel's Rule. Examples of arylgroups are phenyl, biphenyl, naphthyl, anthracenyl, phenanthrenyl, andthe like, as further illustrated below.

“Heteroaryl” means an optionally substituted aromatic monocyclic ring orpolycarbocyclic fused ring system wherein at least one ring complieswith Hückel's Rule, has the specified number of ring atoms, and thatring contains at least one heteratom independently selected from N, Oand S. Examples of “heteroaryl” groups are given herein below.

The term “optionally” means that the subsequently described event(s) mayor may not occur, and includes both event(s), which occur, and eventsthat do not occur.

Herein, the term “pharmaceutically-acceptable salts” refers to saltsthat retain the desired biological activity of the subject compound andexhibit minimal undesired toxicological effects. Thesepharmaceutically-acceptable salts may be prepared in situ during thefinal isolation and purification of the compound, or by separatelyreacting the purified compound in its free acid or free base form with asuitable base or acid, respectively.

Pharmaceutical Formulations

While it is possible that, the compound of the present invention, aswell as pharmaceutically acceptable salts and solvates thereof, may beadministered as the raw chemical, it is also possible to present theactive ingredient as a pharmaceutical composition. Accordingly,embodiments of the invention further provide pharmaceuticalcompositions, which include therapeutically effective amounts of acompound of Formula (I), or Compound A, or Compound B and one or morepharmaceutically acceptable carriers, diluents, or excipients. Thecarrier(s), diluent(s) or excipient(s) must be acceptable in the senseof being compatible with the other ingredients of the formulation andnot deleterious to the recipient thereof. In accordance with anotheraspect of the invention there is also provided a process for thepreparation of a pharmaceutical formulation including admixing acompound of Formula I, Compound A, or Compound B with one or morepharmaceutically acceptable carriers, diluents or excipients.

Pharmaceutical formulations may be presented in unit dose formscontaining a predetermined amount of active ingredient per unit dose.Such a unit may contain, for example, 0.5 mg to 1 g, preferably 1 mg to800 mg, of a compound of the formula (I) depending on the conditionbeing treated, the route of administration and the age, weight andcondition of the patient. Preferred unit dosage formulations are thosecontaining a daily dose or sub-dose, as herein above recited, or anappropriate fraction thereof, of an active ingredient. Furthermore, suchpharmaceutical formulations may be prepared by any of the methods wellknown by one of skill in the art, e.g. in the pharmacy art

Pharmaceutical formulations may be adapted for administration by anyappropriate route, for example by the oral (including buccal orsublingual), rectal, nasal, topical (including buccal, sublingual ortransdermal), vaginal or parenteral (including subcutaneous,intramuscular, intravenous or intradermal) route. Such formulations maybe prepared by any method known in the art of pharmacy, for example bybringing into association the active ingredient with the carrier(s) orexcipient(s).

Pharmaceutical formulations adapted for oral administration may bepresented as discrete units such as capsules or tablets; powders orgranules; solutions or suspensions in aqueous or non-aqueous liquids;edible foams or whips; or oil-in-water liquid emulsions or water-in-oilliquid emulsions.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Powders are prepared by comminuting thecompound to a suitable fine size and mixing with a similarly comminutedpharmaceutical carrier such as an edible carbohydrate, as, for example,starch or mannitol. Flavoring, preservative, dispersing and coloringagent can also be present.

Capsules are made by preparing a powder mixture as described above, andfilling formed gelatin sheaths. Glidants and lubricants such ascolloidal silica, talc, magnesium stearate, calcium stearate or solidpolyethylene glycol can be added to the powder mixture before thefilling operation. A disintegrating or solubilizing agent such asagar-agar, calcium carbonate or sodium carbonate can also be added toimprove the availability of the medicament when the capsule is ingested.

Moreover, when desired or necessary, suitable binders, lubricants,disintegrating agents and coloring agents can also be incorporated intothe mixture. Suitable binders include starch, gelatin, natural sugarssuch as glucose or beta-lactose, corn sweeteners, natural and syntheticgums such as acacia, tragacanth or sodium alginate,carboxymethylcellulose, polyethylene glycol, waxes and the like.Lubricants used in these dosage forms include sodium oleate, sodiumstearate, magnesium stearate, sodium benzoate, sodium acetate, sodiumchloride and the like. Disintegrators include, without limitation,starch, methyl cellulose, agar, bentonite, xanthan gum and the like.Tablets are formulated, for example, by preparing a powder mixture,granulating or slugging, adding a lubricant and disintegrant andpressing into tablets. A powder mixture is prepared by mixing thecompound, suitably comminuted, with a diluent or base as describedabove, and optionally, with a binder such as carboxymethylcellulose, analiginate, gelatin, or polyvinyl pyrrolidone, a solution retardant suchas paraffin, a resorption accelerator such as a quaternary salt and/oran absorption agent such as bentonite, kaolin or dicalcium phosphate.The powder mixture can be granulated by wetting with a binder such assyrup, starch paste, acadia mucilage or solutions of cellulosic orpolymeric materials and forcing through a screen. As an alternative togranulating, the powder mixture can be run through the tablet machineand the result is imperfectly formed slugs broken into granules. Thegranules can be lubricated to prevent sticking to the tablet formingdies by means of the addition of stearic acid, a stearate salt, talc ormineral oil. The lubricated mixture is then compressed into tablets. Thecompounds of the present invention can also be combined with a freeflowing inert carrier and compressed into tablets directly without goingthrough the granulating or slugging steps. A clear or opaque protectivecoating consisting of a sealing coat of shellac, a coating of sugar orpolymeric material and a polish coating of wax can be provided.Dyestuffs can be added to these coatings to distinguish different unitdosages.

Oral fluids such as solution, syrups and elixirs can be prepared indosage unit form so that a given quantity contains a predeterminedamount of the compound. Syrups can be prepared by dissolving thecompound in a suitably flavored aqueous solution, while elixirs areprepared through the use of a non-toxic alcoholic vehicle. Suspensionscan be formulated by dispersing the compound in a non-toxic vehicle.Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols andpolyoxy ethylene sorbitol ethers, preservatives, flavor additives suchas peppermint oil or natural sweeteners or saccharin or other artificialsweeteners, and the like can also be added.

Where appropriate, dosage unit formulations for oral administration canbe microencapsulated. The formulation can also be prepared to prolong orsustain the release as for example by coating or embedding particulatematerial in polymers, wax or the like.

Dosage unit forms can also be in the form for i.v. delivery, of whichone of skill in the art is capable of providing.

Dosage unit forms, e.g. for i.v. delivery, can also be in the form ofliposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles and multilamellar vesicles. Liposomes can be formedfrom a variety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines or other forms familiar to one of skill in the art.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations may include other agents conventionalin the art having regard to the type of formulation in question, forexample those suitable for oral administration may include flavouringagents.

A therapeutically effective amount of a compound of formula (I) or apharmaceutically acceptable salt thereof will depend upon a number offactors including, for example, the age and weight of the animal, theprecise condition requiring treatment and its severity, the nature ofthe formulation, and the route of administration, and will ultimately beat the discretion of the attendant physician or veterinarian. However,an effective amount of a compound of formula (I) or a salt thereof forthe treatment of a cancerous condition such as those described hereinwill generally be in the range of 0.1 to 100 mg/kg body weight ofrecipient (mammal) per day and more usually in the range of 1 to 12mg/kg body weight per day. Thus, for a 70 kg adult mammal, the actualamount per day would usually be from 70 to 840 mg and this amount may begiven in a single dose per day or more usually in a number (such as two,three, four, five or six) of sub-doses per day such that the total dailydose is the same. An effective amount of a salt or solvate thereof maybe determined as a proportion of the effective amount of the compound offormula (I) per se. It is envisaged that similar dosages would beappropriate for treatment of the other conditions referred to above.

The amount of administered or prescribed compound according to theseaspects of the present invention will depend upon a number of factorsincluding, for example, the age and weight of the patient, the precisecondition requiring treatment, the severity of the condition, the natureof the formulation, and the route of administration. Ultimately, theamount will be at the discretion of the attendant physician.

Combinations and Additional Anti-Neoplastic Agents

In certain embodiments, the methods of the present invention furthercomprise administering one or more additional anti-neoplastic agents.

When an EZH2 inhibitor such as, but not limited to, Formula I, CompoundA, or Compound B, is administered for the treatment of cancer, the term“co-administering” and derivatives thereof as used herein is meanteither simultaneous administration or any manner of separate sequentialadministration of an EZH2 inhibiting compound, as described herein, anda further active ingredient or ingredients, known to be useful in thetreatment of cancer, including chemotherapy and radiation treatment. Theterm further active ingredient or ingredients, as used herein, includesany compound or therapeutic agent known to or that demonstratesadvantageous properties when administered to a patient in need oftreatment for cancer. If the administration is not simultaneous, thecompounds are administered in a close time proximity to each other.Furthermore, it does not matter if the compounds are administered in thesame dosage form, e.g. one compound may be administered topically orintraveneously (i.v.) and another compound may be administered orally.

Typically, any anti-neoplastic agent that has activity versus asusceptible tumor or cancer (e.g. lymphoma) being treated may beco-administered in the treatment of cancer in the present invention.Examples of such agents can be found in Cancer Principles and Practice fOncology by V. T. Devita and S. Hellman (editors), 6^(th) edition (Feb.15, 2001), Lippincott Williams & Wilkins Publishers. A person ofordinary skill in the art would be able to discern which combinations ofagents would be useful based on the particular characteristics of thedrugs and the cancer involved. Typical anti-neoplastic agents useful inthe present invention include, but are not limited to, any treatment forlymphoma, such as: R-CHOP, the five component treatment fornon-Hodgkin's lymphoma, comprising: Rituximab, Cyclophosphamide, a DNAalkylating cross-linking agent; Hydroxydaunorubicin (i.e. doxorubicin orAdriamycin), a DNA intercalating agent; Oncovin (vincristine), whichinhibits cell division by binding to the protein tubulin, and thecorticosteroids Prednisone or prednisolone; CHOP, R-CVP (similar toR-CHOP, comprises rituximab, cyclophosphamide, vincristine, andprednisolone/prednisone), CVP; bortezomib; bendamustin; alemtuzumab; andradioimmunotherapy (e. ibritumomab (Zevalin), tositumomab (Bexxar)).

Other typical anti-neoplastic agents useful in the present inventioninclude, but are not limited to. Class I and Class II histonedeacetylase (HDAC) inhibitors (e.g., vorinostat), DNA methylaseinhibitors (e.g. decitabine or azacitidine), histone acetyltransferase(HAT) inhibitors (e.g. p300 and PCAF inhibitors), anti-microtubuleagents such as diterpenoids and vinca alkaloids; platinum coordinationcomplexes; alkylating agents such as nitrogen mustards,oxazaphosphorines, alkylsulfonates, nitrosoureas, and triazenes;antibiotic agents such as anthracyclins, actinomycins and bleomycins;topoisomerase II inhibitors such as epipodophyllotoxins; antimetabolitessuch as purine and pyrimidine analogues and anti-folate compounds;topoisomerase I inhibitors such as camptothecins; hormones and hormonalanalogues; signal transduction pathway inhibitors; non-receptor tyrosinekinase angiogenesis inhibitors; immunotherapeutic agents; proapoptoticagents; and cell cycle signaling inhibitors.

Examples of a further active ingredient or ingredients for use incombination or co-administered with the present EZH2 inhibitingcompounds are chemotherapeutic agents.

Anti-microtubule or anti-mitotic agents are phase specific agents activeagainst the microtubules of tumor cells during M or the mitosis phase ofthe cell cycle. Examples of anti-microtubule agents include, but are notlimited to, diterpenoids and vinca alkaloids.

Diterpenoids, which are derived from natural sources, are phase specificanti-cancer agents that operate at the G2/M phases of the cell cycle. Itis believed that the diterpenoids stabilize the β-tubulin subunit of themicrotubules, by binding with this protein. Disassembly of the proteinappears then to be inhibited with mitosis being arrested and cell deathfollowing. Examples of diterpenoids include, but are not limited to,paclitaxel and its analog docetaxel.

Paclitaxel, 5β,20-epoxy-1,2α,4,7β,10β,13α-hexa-hydroxytax-11-en-9-one4,10-diacetate 2-benzoate 13-ester with (2R,3S)—N-benzoyl-3-phenylisoserine; is a natural diterpene product isolatedfrom the Pacific yew tree Taxus brevifolia and is commercially availableas an injectable solution TAXOL®. It is a member of the taxane family ofterpenes. It was first isolated in 1971 by Wani et al. J. Am. Chem,Soc., 93:2325. 1971), who characterized its structure by chemical andX-ray crystallographic methods. One mechanism for its activity relatesto paclitaxel's capacity to bind tubulin, thereby inhibiting cancer cellgrowth. Schiff et al., Proc. Natl, Acad, Sci. USA, 77:1561-1565 (1980);Schiff et al., Nature, 277:665-667 (1979); Kumar, J. Biol, Chem, 256:10435-10441 (1981). For a review of synthesis and anticancer activity ofsome paclitaxel derivatives see: D. G. I. Kingston et al., Studies inOrganic Chemistry vol. 26, entitled “New trends in Natural ProductsChemistry 1986”, Attaur-Rahman, P. W. Le Quesne, Eds. (Elsevier,Amsterdam, 1986) pp 219-235.

Paclitaxel has been approved for clinical use in the treatment ofrefractory ovarian cancer in the United States (Markman et al., YaleJournal of Biology and Medicine, 64:583, 1991; McGuire et al., Ann.Intem, Med., 111:273, 1989) and for the treatment of breast cancer(Holmes et al., J. Nat. Cancer Inst., 83:1797, 1991.) It is a potentialcandidate for treatment of neoplasms in the skin (Einzig et. al., Proc.Am. Soc. Clin. Oncol., 20:46) and head and neck carcinomas (Forastireet. al., Sem. Oncol., 20:56, 1990). The compound also shows potentialfor the treatment of polycystic kidney disease (Woo et. al., Nature,368:750. 1994), lung cancer and malaria. Treatment of patients withpaclitaxel results in bone marrow suppression (multiple cell lineages,Ignoff, R. J. et. al, Cancer Chemotherapy Pocket Guide. 1998) related tothe duration of dosing above a threshold concentration (50 nM) (Kearns,C. M. et. al., Seminars in Oncology, 3(6) p. 16-23, 1995).

Docetaxel, (2R,3 S)—N-carboxy-3-phenylisoserine, N-tert-butyl ester,13-ester with 5β-20-epoxy-1,2α,4,7β,10β,13α-hexahydroxytax-11-en-9-one4-acetate 2-benzoate, trihydrate; is commercially available as aninjectable solution as TAXOTERE®. Docetaxel is indicated for thetreatment of breast cancer. Docetaxel is a semisynthetic derivative ofpaclitaxel q.v., prepared using a natural precursor,10-deacetyl-baccatin III, extracted from the needle of the European Yewtree. The dose limiting toxicity of docetaxel is neutropenia.

Vinca alkaloids are phase specific anti-neoplastic agents derived fromthe periwinkle plant. Vinca alkaloids act at the M phase (mitosis) ofthe cell cycle by binding specifically to tubulin. Consequently, thebound tubulin molecule is unable to polymerize into microtubules.Mitosis is believed to be arrested in metaphase with cell deathfollowing. Examples of vinca alkaloids include, but are not limited to,vinblastine, vincristine, and vinorelbine.

Vinblastine, vincaleukoblastine sulfate, is commercially available asVELBAN® as an injectable solution. Although, it has possible indicationas a second line therapy of various solid tumors, it is primarilyindicated in the treatment of testicular cancer and various lymphomasincluding Hodgkin's Disease; and lymphocytic and histiocytic lymphomas.Myelosuppression is the dose limiting side effect of vinblastine.

Vincristine, vincaleukoblastine, 22-oxo-, sulfate, is commerciallyavailable as ONCOVIN® as an injectable solution. Vincristine isindicated for the treatment of acute leukemias and has also found use intreatment regimens for Hodgkin's and non-Hodgkin's malignant lymphomas.Alopecia and neurologic effects are the most common side effect ofvincristine and to a lesser extent myelosupression and gastrointestinalmucositis effects occur.

Vinorelbine, 3′,4′-didehydro-4′-deoxy-C′-norvincaleukoblastine[R—(R*,R*)-2,3-dihydroxybutanedioate (1:2)(salt)], commerciallyavailable as an injectable solution of vinorelbine tartrate(NAVELBINE®), is a semisynthetic vinca alkaloid. Vinorelbine isindicated as a single agent or in combination with otherchemotherapeutic agents, such as cisplatin, in the treatment of varioussolid tumors, particularly non-small cell lung, advanced breast, andhormone refractory prostate cancers. Myelosuppression is the most commondose limiting side effect of vinorelbine.

Platinum coordination complexes are non-phase specific anti-canceragents, which are interactive with DNA. The platinum complexes entertumor cells, undergo, aquation and form intra- and interstrandcrosslinks with DNA causing adverse biological effects to the tumor.Examples of platinum coordination complexes include, but are not limitedto, cisplatin and carboplatin.

Cisplatin, cis-diamminedichloroplatinum, is commercially available asPLATINOL® as an injectable solution. Cisplatin is primarily indicated inthe treatment of metastatic testicular and ovarian cancer and advancedbladder cancer. The primary dose limiting side effects of cisplatin arenephrotoxicity, which may be controlled by hydration and diuresis, andototoxicity.

Carboplatin, platinum, diammine[1,1-cyclobutane-dicarboxylate(2-)—O,O′], is commercially available asPARAPLATIN® as an injectable solution. Carboplatin is primarilyindicated in the first and second line treatment of advanced ovariancarcinoma. Bone marrow suppression is the dose limiting toxicity ofcarboplatin.

Alkylating agents are non-phase anti-cancer specific agents and strongelectrophiles. Typically, alkylating agents form covalent linkages, byalkylation, to DNA through nucleophilic moieties of the DNA moleculesuch as phosphate, amino, sulfhydryl, hydroxyl, carboxyl, and imidazolegroups. Such alkylation disrupts nucleic acid function leading to celldeath. Examples of alkylating agents include, but are not limited to,nitrogen mustards such as cyclophosphamide, melphalan, and chlorambucil;alkyl sulfonates such as busulfan; nitrosoureas such as carmustine; andtriazenes such as dacarbazine.

Cyclophosphamide,2-[bis(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxidemonohydrate, is commercially available as an injectable solution ortablets as CYTOXAN®. Cyclophosphamide is indicated as a single agent orin combination with other chemotherapeutic agents, in the treatment ofmalignant lymphomas, multiple myeloma, and leukemias. Alopecia, nausea,vomiting and leukopenia are the most common dose limiting side effectsof cyclophosphamide.

Melphalan, 4-[bis(2-chloroethyl)amino]-L-phenylalanine, is commerciallyavailable as an injectable solution or tablets as ALKERAN®. Melphalan isindicated for the palliative treatment of multiple myeloma andnon-resectable epithelial carcinoma of the ovary. Bone marrowsuppression is the most common dose limiting side effect of melphalan.

Chlorambucil, 4-[bis(2-chloroethyl)amino]benzenebutanoic acid, iscommercially available as LEUKERAN® tablets. Chlorambucil is indicatedfor the palliative treatment of chronic lymphatic leukemia, andmalignant lymphomas such as lymphosarcoma, giant follicular lymphoma,and Hodgkin's disease. Bone marrow suppression is the most common doselimiting side effect of chlorambucil.

Busulfan, 1,4-butanediol dimethanesulfonate, is commercially availableas MYLERAN® TABLETS. Busulfan is indicated for the palliative treatmentof chronic myelogenous leukemia. Bone marrow suppression is the mostcommon dose limiting side effects of busulfan.

Carmustine, 1,3-[bis(2-chloroethyl)-1-nitrosourea, is commerciallyavailable as single vials of lyophilized material as BiCNU®. Carmustineis indicated for the palliative treatment as a single agent or incombination with other agents for brain tumors, multiple myeloma,Hodgkin's disease, and non-Hodgkin's lymphomas. Delayed myelosuppressionis the most common dose limiting side effects of carmustine.

Dacarbazine, 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide, iscommercially available as single vials of material as DTIC-Dome®.Dacarbazine is indicated for the treatment of metastatic malignantmelanoma and in combination with other agents for the second linetreatment of Hodgkin's Disease. Nausea, vomiting, and anorexia are themost common dose limiting side effects of dacarbazine.

Antibiotic anti-neoplastics are non-phase specific agents, which bind orintercalate with DNA. Typically, such action results in stable DNAcomplexes or strand breakage, which disrupts ordinary function of thenucleic acids leading to cell death. Examples of antibioticanti-neoplastic agents include, but are not limited to, actinomycinssuch as dactinomycin, anthrocyclins such as daunorubicin anddoxorubicin; and bleomycins.

Dactinomycin, also know as Actinomycin D, is commercially available ininjectable form as COSMEGEN®. Dactinomycin is indicated for thetreatment of Wilm's tumor and rhabdomyosarcoma. Nausea, vomiting, andanorexia are the most common dose limiting side effects of dactinomycin.

Daunorubicin,(8S-cis-)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12naphthacenedione hydrochloride, is commercially available as a liposomalinjectable form as DAUNOXOME® or as an injectable as CERUBIDINE®.Daunorubicin is indicated for remission induction in the treatment ofacute nonlymphocytic leukemia and advanced HIV associated Kaposi'ssarcoma. Myelosuppression is the most common dose limiting side effectof daunorubicin.

Doxorubicin, (8S,10S)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-8-glycoloyl,7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12 naphthacenedionehydrochloride, is commercially available as an injectable form as RUBEX®or ADRIAMYCIN RDF®. Doxorubicin is primarily indicated for the treatmentof acute lymphoblastic leukemia and acute myeloblastic leukemia, but isalso a useful component in the treatment of some solid tumors andlymphomas. Myelosuppression is the most common dose limiting side effectof doxorubicin.

Bleomycin, a mixture of cytotoxic glycopeptide antibiotics isolated froma strain of Streptomyces verticillus, is commercially available asBLENOXANE®. Bleomycin is indicated as a palliative treatment, as asingle agent or in combination with other agents, of squamous cellcarcinoma, lymphomas, and testicular carcinomas. Pulmonary and cutaneoustoxicities are the most common dose limiting side effects of bleomycin.

Topoisomerase II inhibitors include, but are not limited to,epipodophyllotoxins.

Epipodophyllotoxins are phase specific anti-neoplastic agents derivedfrom the mandrake plant. Epipodophyllotoxins typically affect cells inthe S and G₂ phases of the cell cycle by forming a ternary complex withtopoisomerase II and DNA causing DNA strand breaks. The strand breaksaccumulate and cell death follows. Examples of epipodophyllotoxinsinclude, but are not limited to, etoposide and teniposide.

Etoposide, 4′-demethyl-epipodophyllotoxin9[4,6-0-(R)-ethylidene-3-D-glucopyranoside], is commercially availableas an injectable solution or capsules as VePESID® and is commonly knownas VP-16. Etoposide is indicated as a single agent or in combinationwith other chemotherapy agents in the treatment of testicular andnon-small cell lung cancers. Myelosuppression is the most common sideeffect of etoposide. The incidence of leucopenia tends to be more severethan thrombocytopenia.

Teniposide, 4′-demethyl-epipodophyllotoxin9[4,6-0-(R)-thenylidene-3-D-glucopyranoside], is commercially availableas an injectable solution as VUMON® and is commonly known as VM-26.Teniposide is indicated as a single agent or in combination with otherchemotherapy agents in the treatment of acute leukemia in children.Myelosuppression is the most common dose limiting side effect ofteniposide. Teniposide can induce both leucopenia and thrombocytopenia.

Antimetabolite neoplastic agents are phase specific anti-neoplasticagents that act at S phase (DNA synthesis) of the cell cycle byinhibiting DNA synthesis or by inhibiting purine or pyrimidine basesynthesis and thereby limiting DNA synthesis. Consequently, S phase doesnot proceed and cell death follows. Examples of antimetaboliteanti-neoplastic agents include, but are not limited to, fluorouracil,methotrexate, cytarabine, mecaptopurine, thioguanine, and gemcitabine.

5-fluorouracil, 5-fluoro-2,4-(1H,3H) pyrimidinedione, is commerciallyavailable as fluorouracil. Administration of 5-fluorouracil leads toinhibition of thymidylate synthesis and is also incorporated into bothRNA and DNA. The result typically is cell death. 5-fluorouracil isindicated as a single agent or in combination with other chemotherapyagents in the treatment of carcinomas of the breast, colon, rectum,stomach and pancreas. Myelosuppression and mucositis are dose limitingside effects of 5-fluorouracil. Other fluoropyrimidine analogs include5-fluoro deoxyuridine (floxuridine) and 5-fluorodeoxyuridinemonophosphate.

Cytarabine, 4-amino-1-β-D-arabinofuranosyl-2 (1H)-pyrimidinone, iscommercially available as CYTOSAR-U® and is commonly known as Ara-C. Itis believed that cytarabine exhibits cell phase specificity at S-phaseby inhibiting DNA chain elongation by terminal incorporation ofcytarabine into the growing DNA chain. Cytarabine is indicated as asingle agent or in combination with other chemotherapy agents in thetreatment of acute leukemia. Other cytidine analogs include5-azacytidine and 2′,2′-difluorodeoxycytidine (gemcitabine). Cytarabineinduces leucopenia, thrombocytopenia, and mucositis.

Mercaptopurine, 1,7-dihydro-6H-purine-6-thione monohydrate, iscommercially available as PURINETHOL®. Mercaptopurine exhibits cellphase specificity at S-phase by inhibiting DNA synthesis by an as of yetunspecified mechanism. Mercaptopurine is indicated as a single agent orin combination with other chemotherapy agents in the treatment of acuteleukemia. Myelosuppression and gastrointestinal mucositis are expectedside effects of mercaptopurine at high doses. A useful mercaptopurineanalog is azathioprine.

Thioguanine, 2-amino-1,7-dihydro-6H-purine-6-thione, is commerciallyavailable as TABLOID®. Thioguanine exhibits cell phase specificity atS-phase by inhibiting DNA synthesis by an as of yet unspecifiedmechanism. Thioguanine is indicated as a single agent or in combinationwith other chemotherapy agents in the treatment of acute leukemia.Myelosuppression, including leucopenia, thrombocytopenia, and anemia, isthe most common dose limiting side effect of thioguanine administration.However, gastrointestinal side effects occur and can be dose limiting.Other purine analogs include pentostatin, erythrohydroxynonyladenine,fludarabine phosphate, and cladribine.

Gemcitabine, 2′-deoxy-2′, 2′-difluorocytidine monohydrochloride(β-isomer), is commercially available as GEMZAR®. Gemcitabine exhibitscell phase specificity at S-phase and by blocking progression of cellsthrough the G1/S boundary. Gemcitabine is indicated in combination withcisplatin in the treatment of locally advanced non-small cell lungcancer and alone in the treatment of locally advanced pancreatic cancer.Myelosuppression, including leucopenia, thrombocytopenia, and anemia, isthe most common dose limiting side effect of gemcitabine administration.

Methotrexate, N-[4[[(2,4-diamino-6-pteridinyl) methyl]methylamino]benzoyl]-L-glutamic acid, is commercially available as methotrexatesodium. Methotrexate exhibits cell phase effects specifically at S-phaseby inhibiting DNA synthesis, repair and/or replication through theinhibition of dyhydrofolic acid reductase which is required forsynthesis of purine nucleotides and thymidylate. Methotrexate isindicated as a single agent or in combination with other chemotherapyagents in the treatment of choriocarcinoma, meningeal leukemia,non-Hodgkin's lymphoma, and carcinomas of the breast, head, neck, ovaryand bladder. Myelosuppression (leucopenia, thrombocytopenia, and anemia)and mucositis are expected side effect of methotrexate administration.

Camptothecins, including, camptothecin and camptothecin derivatives areavailable or under development as Topoisomerase I inhibitors.Camptothecins cytotoxic activity is believed to be related to itsTopoisomerase I inhibitory activity. Examples of camptothecins include,but are not limited to irinotecan, topotecan, and the various opticalforms of 7-(4-methylpiperazino-methylene)-10,11,-ethylenedioxy-20-camptothecin described below.

Irinotecan HCl, (4S)-4,11-diethyl-4-hydroxy-9-[(4-piperidinopiperidino)carbonyloxy]-1H-pyrano[3′,4′,6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione hydrochloride, is commercially available as the injectablesolution CAMPTOSAR®.

Irinotecan is a derivative of camptothecin which binds, along with itsactive metabolite SN-38, to the topoisomerase I-DNA complex. It isbelieved that cytotoxicity occurs as a result of irreparable doublestrand breaks caused by interaction of the topoisomerase I: DNA:irintecan or SN-38 ternary complex with replication enzymes. Irinotecanis indicated for treatment of metastatic cancer of the colon or rectum.The dose limiting side effects of irinotecan HCl are myelosuppression,including neutropenia, and G₁ effects, including diarrhea.

Topotecan HCl,(S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3′,4′,6,7]indolizino[1,2-b]quinoline-3,14-(4H,12H)-dionemonohydrochloride, is commercially available as the injectable solutionHYCAMTIN®. Topotecan is a derivative of camptothecin which binds to thetopoisomerase I-DNA complex and prevents religation of singles strandbreaks caused by Topoisomerase I in response to torsional strain of theDNA molecule. Topotecan is indicated for second line treatment ofmetastatic carcinoma of the ovary and small cell lung cancer. The doselimiting side effect of topotecan HCl is myelosuppression, primarilyneutropenia.

Also of interest, is the camptothecin derivative of formula F following,currently under development, including the racemic mixture (R,S) form aswell as the R and S enantiomers:

known by the chemical name“7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20(R,S)-camptothecin(racemic mixture) or“7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20(R)-camptothecin(R enantiomer) or“7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20(S)-camptothecin(S enantiomer). Such compound as well as related compounds aredescribed, including methods of making, in U.S. Pat. Nos. 6,063,923;5,342,947; 5,559,235; 5,491,237 and pending U.S. patent application Ser.No. 08/977,217 filed Nov. 24, 1997.

Hormones and hormonal analogues are useful compounds for treatingcancers in which there is a relationship between the hormone(s) andgrowth and/or lack of growth of the cancer. Examples of hormones andhormonal analogues useful in cancer treatment include, but are notlimited to, adrenocorticosteroids such as prednisone and prednisolonewhich are useful in the treatment of malignant lymphoma and acuteleukemia in children; aminoglutethimide and other aromatase inhibitorssuch as anastrozole, letrazole, vorazole, and exemestane useful in thetreatment of adrenocortical carcinoma and hormone dependent breastcarcinoma containing estrogen receptors; progestrins such as megestrolacetate useful in the treatment of hormone dependent breast cancer andendometrial carcinoma; estrogens, androgens, and anti-androgens such asflutamide, nilutamide, bicalutamide, cyproterone acetate and5α-reductases such as finasteride and dutasteride, useful in thetreatment of prostatic carcinoma and benign prostatic hypertrophy;anti-estrogens such as tamoxifen, toremifene, raloxifene, droloxifene,iodoxyfene, as well as selective estrogen receptor modulators (SERMS)such those described in U.S. Pat. Nos. 5,681,835, 5,877,219, and6,207,716, useful in the treatment of hormone dependent breast carcinomaand other susceptible cancers; and gonadotropin-releasing hormone (GnRH)and analogues thereof which stimulate the release of leutinizing hormone(LH) and/or follicle stimulating hormone (FSH) for the treatmentprostatic carcinoma, for instance, LHRH agonists and antagagonists suchas goserelin acetate and luprolide.

Letrozole (trade name Femara) is an oral non-steroidal aromataseinhibitor for the treatment of hormonally-responsive breast cancer aftersurgery. Estrogens are produced by the conversion of androgens throughthe activity of the aromatase enzyme. Estrogens then bind to an estrogenreceptor, which causes cells to divide. Letrozole prevents the aromatasefrom producing estrogens by competitive, reversible binding to the hemeof its cytochrome P450 unit. The action is specific, and letrozole doesnot reduce production of mineralo- or corticosteroids.

Signal transduction pathway inhibitors are those inhibitors, which blockor inhibit a chemical process which evokes an intracellular change. Asused herein this change is cell proliferation or differentiation. Signaltransduction inhibitors useful in the present invention includeinhibitors of receptor tyrosine kinases, non-receptor tyrosine kinases,SH2/SH3 domain blockers, serine/threonine kinases, phosphotidylinositol-3 kinases, myo-inositol signaling, and Ras oncogenes.

Several protein tyrosine kinases catalyse the phosphorylation ofspecific tyrosyl residues in various proteins involved in the regulationof cell growth. Such protein tyrosine kinases can be broadly classifiedas receptor or non-receptor kinases.

Receptor tyrosine kinases are transmembrane proteins having anextracellular ligand binding domain, a transmembrane domain, and atyrosine kinase domain. Receptor tyrosine kinases are involved in theregulation of cell growth and are generally termed growth factorreceptors. Inappropriate or uncontrolled activation of many of thesekinases, i.e. aberrant kinase growth factor receptor activity, forexample by over-expression or mutation, has been shown to result inuncontrolled cell growth. Accordingly, the aberrant activity of suchkinases has been linked to malignant tissue growth. Consequently,inhibitors of such kinases could provide cancer treatment methods.Growth factor receptors include, for example, epidermal growth factorreceptor (EGFr), platelet derived growth factor receptor (PDGFr), erbB2,erbB4, vascular endothelial growth factor receptor (VEGFr), tyrosinekinase with immunoglobulin-like and epidermal growth factor homologydomains (TIE-2), insulin growth factor-I (IGFI) receptor, macrophagecolony stimulating factor (cfms), BTK, ckit, cmet, fibroblast growthfactor (FGF) receptors, Trk receptors (TrkA, TrkB, and TrkC), ephrin(eph) receptors, and the RET protooncogene. Several inhibitors of growthreceptors are under development and include ligand antagonists,antibodies, tyrosine kinase inhibitors and anti-sense oligonucleotides.Growth factor receptors and agents that inhibit growth factor receptorfunction are described, for instance, in Kath, John C., Exp. Opin. Ther.Patents (2000) 10(6):803-818; Shawver et al DDT Vol 2, No. 2 Feb. 1997;and Lofts, F. J. et al, “Growth factor receptors as targets”, NewMolecular Targets for Cancer Chemotherapy, ed. Workman, Paul and Kerr,David, CRC press 1994, London.

Tyrosine kinases, which are not growth factor receptor kinases aretermed non-receptor tyrosine kinases. Non-receptor tyrosine kinasesuseful in the present invention, which are targets or potential targetsof anti-cancer drugs, include cSrc, Lck, Fyn, Yes, Jak, cAbl, FAK (Focaladhesion kinase), Brutons tyrosine kinase, and Bcr-Abl. Suchnon-receptor kinases and agents which inhibit non-receptor tyrosinekinase function are described in Sinh, S. and Corey, S. J., (1999)Journal of Hematotherapy and Stem Cell Research 8 (5): 465-80; andBolen, J. B., Brugge, J. S., (1997) Annual review of Immunology. 15:371-404.

SH2/SH3 domain blockers are agents that disrupt SH2 or SH3 domainbinding in a variety of enzymes or adaptor proteins including, PI3-K p85subunit, Src family kinases, adaptor molecules (Shc, Crk, Nck, Grb2) andRas-GAP. SH2/SH3 domains as targets for anti-cancer drugs are discussedin Smithgall, T. E. (1995), Journal of Pharmacological and ToxicologicalMethods. 34(3) 125-32.

Inhibitors of Serine/Threonine Kinases including MAP kinase cascadeblockers which include blockers of Raf kinases (rafk), Mitogen orExtracellular Regulated Kinase (MEKs), and Extracellular RegulatedKinases (ERKs); and Protein kinase C family member blockers includingblockers of PKCs (alpha, beta, gamma, epsilon, mu, lambda, iota, zeta).IkB kinase family (IKKa, IKKb), PKB family kinases, AKT kinase familymembers, and TGF beta receptor kinases. Such Serine/Threonine kinasesand inhibitors thereof are described in Yamamoto, T., Taya, S.,Kaibuchi, K., (1999), Journal of Biochemistry. 126 (5) 799-803; Brodt,P, Samani, A., and Navab, R. (2000), Biochemical Pharmacology, 60.1101-1107; Massague, J., Weis-Garcia, F. (1996) Cancer Surveys.27:41-64; Philip, P. A., and Harris, A. L. (1995), Cancer Treatment andResearch. 78: 3-27, Lackey, K. et al Bioorganic and Medicinal ChemistryLetters, (10), 2000, 223-226; U.S. Pat. No. 6,268,391; andMartinez-Iacaci, L., et al, Int. J. Cancer (2000), 88(1), 44-52.

Inhibitors of Phosphotidyl inositol-3 Kinase family members includingblockers of PI3-kinase, ATM, DNA-PK, and Ku are also useful in thepresent invention. Such kinases are discussed in Abraham, R. T. (1996),Current Opinion in Immunology. 8 (3) 412-8; Canman, C. E., Lim, D. S.(1998), Oncogene 17 (25) 3301-3308; Jackson, S. P. (1997), InternationalJournal of Biochemistry and Cell Biology. 29 (7):935-8; and Zhong, H. etal, Cancer res, (2000) 60(6), 1541-1545.

Also useful in the present invention are Myo-inositol signalinginhibitors such as phospholipase C blockers and Myoinositol analogues.Such signal inhibitors are described in Powis, G., and Kozikowski A.,(1994) New Molecular Targets for Cancer Chemotherapy ed., Paul Workmanand David Kerr, CRC press 1994, London.

Another group of signal transduction pathway inhibitors are inhibitorsof Ras Oncogene. Such inhibitors include inhibitors offarnesyltransferase, geranyl-geranyl transferase, and CAAX proteases aswell as anti-sense oligonucleotides, ribozymes and immunotherapy. Suchinhibitors have been shown to block ras activation in cells containingwild type mutant ras, thereby acting as antiproliferation agents. Rasoncogene inhibition is discussed in Scharovsky, O. G., Rozados, V. R.,Gervasoni, S. I. Matar, P. (2000), Journal of Biomedical Science. 7(4)292-8; Ashby, M. N. (1998), Current Opinion in Lipidology. 9 (2) 99,—102; and Bennett, C. F. and Cowsert, L. M. BioChim. Biophys. Acta,(1999) 1489(1):19-30.

As mentioned above, antibody antagonists to receptor kinase ligandbinding may also serve as signal transduction inhibitors. This group ofsignal transduction pathway inhibitors includes the use of humanizedantibodies to the extracellular ligand binding domain of receptortyrosine kinases. For example Imclone C225 EGFR specific antibody (seeGreen, M. C. et al, Monoclonal Antibody Therapy for Solid Tumors, CancerTreat. Rev., (2000), 26(4), 269-286); Herceptin® erbB2 antibody (seeTyrosine Kinase Signalling in Breast cancer:erbB Family ReceptorTyrosine Kinases, Breast cancer Res., 2000, 2(3), 176-183); and 2CBVEGFR2 specific antibody (see Brekken, R. A. et al, Selective Inhibitionof VEGFR2 Activity by a monoclonal Anti-VEGF antibody blocks tumorgrowth in mice, Cancer Res. (2000) 60, 5117-5124).

Non-receptor kinase angiogenesis inhibitors may also find use in thepresent invention. Inhibitors of angiogenesis related VEGFR and TIE2 arediscussed above in regard to signal transduction inhibitors (bothreceptors are receptor tyrosine kinases). Angiogenesis in general islinked to erbB2/EGFR signaling since inhibitors of erbB2 and EGFR havebeen shown to inhibit angiogenesis, primarily VEGF expression. Thus, thecombination of an erbB2/EGFR inhibitor with an inhibitor of angiogenesismakes sense. Accordingly, non-receptor tyrosine kinase inhibitors may beused in combination with the EGFR/erbB2 inhibitors of the presentinvention. For example, anti-VEGF antibodies, which do not recognizeVEGFR (the receptor tyrosine kinase), but bind to the ligand; smallmolecule inhibitors of integrin (alpha_(v) beta₃) that will inhibitangiogenesis; endostatin and angiostatin (non-RTK) may also prove usefulin combination with the disclosed erb family inhibitors. (See Bruns C Jet al (2000), Cancer Res., 60: 2926-2935; Schreiber A B, Winkler M E,and Derynck R. (1986), Science, 232: 1250-1253; Yen L et al. (2000),Oncogene 19: 3460-3469).

Pazopanib which commercially available as VOTRIENT® is a tyrosine kinaseinhibitor (TKI). Pazopanib is presented as the hydrochloride salt, withthe chemical name5-[[4-[(2,3-dimethyl-2H-indazol-6-yl)methylamino]-2-pyrimidinyl]amino]-2-methylbenzenesulfonamidemonohydrochloride. Pazoponib is approved for treatment of patients withadvanced renal cell carcinoma.

Bevacisumab which is commercially available as AVASTIN® is a humanizedmonoclonal antibody that blocks VEGF-A. AVASTIN® is approved form thetreatment of various cancers including colorectal, lung, breast, kidney,and glioblastomas.

mTOR inhibitors include but are not limited to rapamycin (FK506) andrapalogs, RAD001 or everolimus (Afinitor), CCI-779 or temsirolimus,AP23573, AZD8055, WYE-354, WYE-600, WYE-687 and Pp121.

Everolimus is sold as Afinitor® by Novartis and is the40-O-(2-hydroxyethyl) derivative of sirolimus and works similarly tosirolimus as an mTOR (mammalian target of rapamycin) inhibitor. It iscurrently used as an immunosuppressant to prevent rejection of organtransplants and treatment of renal cell cancer. Much research has alsobeen conducted on everolimus and other mTOR inhibitors for use in anumber of cancers. It has the following chemical structure (formula V)and chemical name:

-   -   dihydroxy-12-[(2R)-1-[(1        S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]propan-2-yl]-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30.3.10.0^(4,9)]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentone.

Bexarotene is sold as Targretin® and is a member of a subclass ofretinoids that selectively activate retinoid X receptors (RXRs). Theseretinoid receptors have biologic activity distinct from that of retinoicacid receptors (RARs). The chemical name is4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl) ethenyl]benzoic acid. Bexarotene is used to treat cutaneous T-cell lymphoma(CTCL, a type of skin cancer) in people whose disease could not betreated successfully with at least one other medication.

Sorafenib marketed as Nexavar® is in a class of medications calledmultikinase inhibitors. Its chemical name is4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methyl-pyridine-2-carboxamide.Sorafenib is used to treat advanced renal cell carcinoma (a type ofcancer that begins in the kidneys). Sorafenib is also used to treatunresectable hepatocellular carcinoma (a type of liver cancer thatcannot be treated with surgery).

Agents used in immunotherapeutic regimens may also be useful incombination with the compounds of formula (I). There are a number ofimmunologic strategies to generate an immune response against erbB2 orEGFR. These strategies are generally in the realm of tumor vaccinations.The efficacy of immunologic approaches may be greatly enhanced throughcombined inhibition of erbB2/EGFR signaling pathways using a smallmolecule inhibitor. Discussion of the immunologic/tumor vaccine approachagainst erbB2/EGFR are found in Reilly R T et al. (2000), Cancer Res.60: 3569-3576; and Chen Y, Hu D, Eling D J, Robbins J, and Kipps T J.(1998), Cancer Res. 58: 1965-1971.

Examples of erbB inhibitors include lapatinib, erlotinib, and gefitinib.Lapatinib,N-(3-chloro-4-{[(3-fluorophenyl)methyl]oxy}phenyl)-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furanyl]-4-quinazolinamine(represented by Formula VI, as illustrated), is a potent, oral,small-molecule, dual inhibitor of erbB-1 and erbB-2 (EGFR and HER2)tyrosine kinases that is approved in combination with capecitabine forthe treatment of HER2-positive metastatic breast cancer.

The free base, HCl salts, and ditosylate salts of the compound offormula (VI) may be prepared according to the procedures disclosed in WO99/35146, published Jul. 15, 1999; and WO 02/02552 published Jan. 10,2002.

Erlotinib,N-(3-ethynylphenyl)-6,7-bis{[2-(methyloxy)ethyl]oxy}-4-quinazolinamineCommercially available under the tradename Tarceva) is represented byformula VII, as illustrated:

The free base and HCl salt of erlotinib may be prepared, for example,according to U.S. Pat. No. 5,747,498, Example 20.

Gefitinib,4-quinazolinamine,N-(3-chloro-4-fluorophenyl)-7-methoxy-6-[3-4-morpholin)propoxy]is represented by formula VIII, as illustrated:

Gefitinib, which is commercially available under the trade name IRESSA®(Astra-Zenenca) is an erbB-1 inhibitor that is indicated as monotherapyfor the treatment of patients with locally advanced or metastaticnon-small-cell lung cancer after failure of both platinum-based anddocetaxel chemotherapies. The free base, HCl salts, and diHCl salts ofgefitinib may be prepared according to the procedures of InternationalPatent Application No. PCT/GB96/00961, filed Apr. 23, 1996, andpublished as WO 96/33980 on Oct. 31, 1996.

Trastuzumab (HEREPTIN®) is a humanized monoclonal antibody that binds tothe HER2 receptor. It original indication is HER2 positive breastcancer.

Cetuximab (ERBITUX®) is a chimeric mouse human antibody that inhibitsepidermal growth factor receptor (EGFR).

Pertuzumab (also called 2C₄, trade name Omnitarg) is a monoclonalantibody. The first of its class in a line of agents called “HERdimerization inhibitors”. By binding to HER2, it inhibits thedimerization of HER2 with other HER receptors, which is hypothesized toresult in slowed tumor growth. Pertuzumab is described in WO01/00245published Jan. 4, 2001.

Rituximab is a chimeric monoclonal antibody which is sold as RITUXAN®and MABTHERA®. Rituximab binds to CD20 on B cells and causes cellapoptosis. Rituximab is administered intravenously and is approved fortreatment of rheumatoid arthritis and B-cell non-Hodgkin's lymphoma.

Ofatumumab is a fully human monoclonal antibody which is sold asARZERRA®. Ofatumumab binds to CD20 on B cells and is used to treatchronic lymphocytic leukemia (CLL; a type of cancer of the white bloodcells) in adults who are refractory to treatment with fludarabine(Fludara) and alemtuzumab (Campath).

Agents used in proapoptotic regimens (e.g., bcl-2 antisenseoligonucleotides) may also be used in the combination of the presentinvention. Members of the Bcl-2 family of proteins block apoptosis.Upregulation of bcl-2 has therefore been linked to chemoresistance.Studies have shown that the epidermal growth factor (EGF) stimulatesanti-apoptotic members of the bcl-2 family (i.e., mcl-1). Therefore,strategies designed to downregulate the expression of bcl-2 in tumorshave demonstrated clinical benefit and are now in Phase II/III trials,namely Genta's G3139 bcl-2 antisense oligonucleotide. Such proapoptoticstrategies using the antisense oligonucleotide strategy for bcl-2 arediscussed in Water J S et al. (2000), J. Clin. Oncol. 18: 1812-1823; andKitada S et al. (1994), Antisense Res. Dev. 4: 71-79.

Cell cycle signaling inhibitors inhibit molecules involved in thecontrol of the cell cycle. A family of protein kinases called cyclindependent kinases (CDKs) and their interaction with a family of proteinstermed cyclins controls progression through the eukaryotic cell cycle.The coordinate activation and inactivation of different cyclin/CDKcomplexes is necessary for normal progression through the cell cycle.Several inhibitors of cell cycle signalling are under development. Forinstance, examples of cyclin dependent kinases, including CDK2, CDK4,and CDK6 and inhibitors for the same are described in, for instance,Rosania et al, Exp. Opin. Ther. Patents (2000) 10(2):215-230.

Any of the cancer treatment methods of the claimed invention may furthercomprise treatment with at least one additional anti-neoplastic agent,such as one selected from the group consisting of anti-microtubuleagents, platinum coordination complexes, alkylating agents, antibioticagents, topoisomerase II inhibitors, antimetabolites, topoisomerase Iinhibitors, hormones and hormonal analogues, signal transduction pathwayinhibitors, non-receptor tyrosine kinase angiogenesis inhibitors,immunotherapeutic agents, proapoptotic agents, and cell cycle signalinginhibitors if one of a mutation in EZH2 at Y641 or A677 or an increasedlevel of H3K27me3 is detected.

EXAMPLES Example 1: Cell Culture and Primary Tumor Specimens

The Pfeiffer DLBCL cell line (ATCC) was maintained in RPMI-1640 media(MediaTech) supplemented with 10% fetal bovine serum (FBS)(Sigma-Aldrich). All other cell line were obtained from either ATCC orDSMZ and maintained in the recommended cell culture media. Genomic DNAfor 41 lymphoma patients was sourced from OriGene (Rockville, Md.). DNAwas extracted from frozen blocks of pathology-verified tumor tissue withhigh tumor content (>40%). Tissues were collected for molecular analysesin accordance with IRB approval and patient informed consent. Detailedclinical characteristics of the tumor specimens are presented in Table2.

Example 2 ELISA-Based Quantitation of Histone H3 and H3K27Me3 Levels

Cell lines were harvested, rinsed with 1×PBS, and centrifuged. Cellpellets were lysed with 0.2N HCl for 30 minutes to extract histones. Theacid insoluble portion was pelleted by centrifugation and thesupernatant was neutralized with neutralization buffer (1M Na₂HPO₄, pH12.5; ActiveMotif) containing protease inhibitors (Roche). Proteinlysates and recombinant H3K27me3 (ActiveMotif) were titrated inneutralized lysis buffer and added to Maxisorp ELISA plates (Nunc) induplicate on each of two plates. Blocking buffer (1% BSA) was added andplates were incubated for 1 hour prior to being washed 4 times withimidazole buffered saline containing Tween-20 (KPL). Plates wereincubated with primary antibodies for H3K27me3 (Cell SignalingTechnology) or total H3 (ActiveMotif), washed, and incubated withsecondary anti-rabbit IgG HRP-linked antibody (Cell SignalingTechnology). Luminata Forte substrate (Millipore) was added 5 minutesbefore quantifying chemiluminescence using an EnVision multi-label platereader (PerkinElmer). H3K27me3 and total H3 levels per cell line werecalculated using the recombinant H3K27me3 signals as a standard controland H3K27me3 values were normalized to total H3 values.

Example 3 Western Blot Analysis of H3K27 Methylation Status

Cell lines were rinsed with PBS and lysed on ice with RIPA buffer(Thermo Scientific) containing protease and phosphatase inhibitors(Roche). Lysates were then sonicated for 15 seconds using a BransonSonifier 150 (setting=2) and protein concentrations were determined witha BCA protein assay (Thermo Scientific). Protein lysates (1-5 □g) werethen denatured and electrophoresed on Bis-Tris SDS-polyacrylamide gels(Invitrogen) before being transferred to PVDF membrane (Invitrogen). Themembranes were blocked with Odyssey blocking buffer (Li-Cor) and probedwith antibodies recognizing EZH2 (BD Transduction Labs), histone H3(Abcam), H3K27me1, H3K27me3 (Cell Signaling Technology), or Actin(Sigma). After washing with PBS containing 0.1% Tween-20 (PBST),membranes were hybridized with fluorescent secondary antibodies(Li-Cor), washed with PBST, and imaged using an Odyssey infrared imagingsystem (Li-Cor).

Example 4 Full-Length Sanger Sequencing of EZH2

Genomic DNA (gDNA) was isolated from cell pellets using the Maxwell 16Cell DNA Purification Kit (Promega). Total RNA was prepared using amodified RNeasy kit procedure (Qiagen) and converted into cDNA using theHiFi First Strand cDNA Synthesis Kit protocol (Roche Diagnostics). PCRreactions were carried out using primers (Table 3) tailed with the M13universal sequencing primer sequences (Integrated DNA Technologies) andHotstarTaq DNA polymerase (Qiagen). DNA (50 ng) was amplified and PCRproducts were purified using AmPure (Agencourt Bioscience). Directsequencing of purified PCR products with M13 primers was performed witha 3730XL Genetic Analyzer (Applied Biosystems) using the v3.1 BigDye-terminator cycle sequencing kit (Applied Biosystems). The sequencingreactions were analyzed and all sequences were assembled and analyzedusing Codon Code Aligner (CodonCode Corporation).

Example 5 Transient Expression of Wild-Type and Mutant EZH2 Proteins inCells

MCF-7 breast cancer cells (3×10⁵; ATCC) were seeded into 6-well tissueculture plates in RPMI-1640 media supplemented with 10% FBS the daybefore transfection. Following the manufacturer's recommendations, 2 μgplasmid DNA and 6 μl Lipofectamine 2000 (Invitrogen) were combined in500 μl Opti-MEM (Invitrogen) and incubated for 20 minutes at roomtemperature before being added to cells. Cells were then incubated at37° C. with 5% CO₂ until harvested for protein lysates and western blotanalysis as described above.

Example 6 Cloning, Expression, and Purification of EZH2 Wild-Type andMutant 5-Member PRC2

Human EED1 (NM_003797), SUZ12 (NP_056170), RbAp48 (RBBP4; NP_005601),and AEBP2 (NP_694939) were cloned into pENTR/TEV/D-TOPO (Invitrogen) andsub-cloned into pDEST8 (Invitrogen). Human EZH2 (NP_001190176) wascloned into pENTR/TEV/D-TOPO and sub-cloned into pDEST8 containing anN-terminal FLAG epitope tag. Human EZH2 in pENTR/TEV/D-TOPO wasmutagenized to introduce single amino acid changes of A677G, Y641N,Y641F, Y641C, Y641H, or Y641S by site-directed mutagenesis (QuikChangeII XL, Agilent Technologies) (Table 4) and sub-cloned into pDEST8containing an N-terminal FLAG epitope tag. The entire coding region ofall mutants was confirmed by double stranded sequencing. For mammalianexpression studies, wild-type human EZH2 was sub-cloned intopIRES2-ZsGreen1 (Clontech). Site-directed mutagenesis was then utilizedas described above to obtain the A677 and Y641 mutants.

Individual baculovirus stocks were generated for expression of EED1,SUZ12, RbAp48, AEBP2, and FLAG-tev-EZH2 using the Bac-to-Bac® system(Invitrogen). All five PRC2 components were co-expressed in Sf9 insectcells by addition of 2×10⁷ baculovirus infected insect cells per 10 Lwave bag for each component. Cells were incubated at 27° C. and cellpaste was harvested between 66-70 hours post-infection.

PRC2 complexes (EZH2, EED, SUZ12, AEBP2, RbAp48) containing flag-taggedwild-type EZH2 or mutant EZH2 (A677G, Y641F, Y641N, Y641S, Y641H, orY641C) were purified from 1 L Sf9 cell paste lysate at 4° C. using 10 mlanti-FLAG M2 resin (Sigma). Resin was packed into a XK26 column andwashed with Buffer A (50 mM Tris HCl pH7.5, 250 mM NaCl, 2 mM DTT). PRC2complex was then eluted with Buffer A containing 100 g/mL FLAG peptide(California Peptide Research). Fractions containing EZH2 were thenpooled and applied to a Superdex 200 column (16/60) (GE Pharmacia)equilibrated with Buffer A. Monomer PRC2 complexes were collected,purity was assessed by SDS-PAGE, and all components and EZH2 mutationswere confirmed by peptide mapping analysis.

Example 7 Biochemical Evaluation of Methyltransferase Activity

Unless stated otherwise, all reagents were obtained from Sigma and wereat a minimum of reagent grade. Custom syntheses of histone H3 peptides(AA21-44; ATKAARKSAPATGGVKKPHRYRPGG[K-Ahx-biotin]-amide) with lysine 27unmodified, mono-methylated, or di-methylated were purchased from21^(st) Century Biochemicals (Marlboro, Mass.). Peptides containedwithin the library were acquired from 21^(st) Century Biochemicals,AnaSpec (Fremont, Calif.) or Alta Bioscience (Birmingham, UK).MicroScint20, streptavidin SPA bead (RPNQ0261), and[³H-]S-adenosyl-methionine (SAM) were purchased from PerkinElmer.

All reactions were evaluated at ambient temperature in assay buffercontaining 50 mM Tris-HCl, (pH8.0), 2 mM MgCl₂, 4 mM DTT, and Tween-20(0.001% for EZH2 peptide activity assays; 0.002% for nucleosome activityassays).

For the peptide assays, EZH2 (20 nM) was added to a 96-well reactionplate (Corning) containing varying concentrations of the H3K27 peptideand [³H-]SAM. Reactions were quenched during the linear portion of theirprogress curves with the addition of 0.1 mM unlabeled SAM. The quenchedreaction mixture was transferred to a 96-well multi-screen HTS filterplate (Millipore MSPHNXB50) that was pre-washed with 0.2 M NH₄HCO₃, pH8.0 (wash buffer 1), and incubated for 30 minutes to allow for capture.Filter plates were then washed with four additional 150 μL aliquots ofwash buffer 1, and allowed to dry. MicroScint20 (60 μL/well) was added,sealed plates were incubated for 30 minutes, and quantitation ofcaptured [³H-]Me-peptide or [³H-]Me-nucleosomes was monitored in aTopCount liquid scintillation counter. Output DPM's were normalized toreaction standards placed onto individual plates following filtration.

For the nucleosome assays, a similar procedure was used with theexception of the filter plates (Millipore MSDEN6B50) and wash buffer (50mM potassium phosphate, pH7.6, wash buffer 2). Mono- and di-nucleosomeswere purified from HeLa cells as described previously (35). Themolecular weight of an individual mono-nucleosome unit within theheterogeneous native nucleosome preparation was estimated based on thecumulative weights of the individual histones [2×H2A (2×14.0 kDa)+2×H2B(2×13.8 kDa)+2×H3 (2×15.2 kDa)+2×H4 (2×1.2 kDa)+200 base pairs dsDNA(200×0.66 kDa)].

Rate data obtained from substrate co-titrations were fitted to Equation(1), which conforms to a sequential kinetic mechanism. For Equation (1),V is the maximal velocity, k_(cat) represents V normalized to enzymeconcentration, A and B represent substrate concentrations, K_(ia) is thedissociation constant for A, and K_(a) and K_(b) are Michaelis constantsfor substrates A and B, respectively.

v=VAB/(K _(ia) K _(b) +K _(a) B+K _(b) A+AB)  (1)

Example 8 Structural Modeling of EZH2

A homology model of wild type EZH2 was built using GLP/EHMT1 bound to anH3K9me2 peptide substrate (PDB ID=2RFI) as a primary template andstructurally compared to other related SET domain containing histonelysine methyltransferases with determined crystal structures. Thehomology model was constructed using the 2009 version of MolecularOperating Environment (MOE) from Chemical Computing Group (CCG).Residues were mutated in MOE and optimal orientations chosen from anexploration of rotamer geometries. For the model of the A677G mutant(FIG. 5C), the low energy rotamer of Y641 was selected after manuallyrotating the di-methylated lysine in an orientation optimal fortri-methylation.

Example 9 Assay Protocol to Evaluate Effects on Proliferation andCalculate Growth IC₅₀s (gIC50s)

Cells were cultured in appropriate medium in flasks to 80-100%confluency. Cells were harvested, counted and plated at a pre-determinedoptimal seeding density ranging from 50 cells per well to 4000 cells perwell depending on the growth characteristics of individual cell lines in384-well tissue culture plates (Greiner #781090). Plates were thenincubated overnight at 37° C., 5% CO₂. Cells were treated with compoundusing a 20 point 1:2 fold dilution scheme with a final concentration of0.15% DMSO. One column per plate contained cells treated with 0.15% DMSOonly, and one column contained no cells to serve as background. Plateswere incubated at 37° C., 5% CO₂ for 6 days. A time zero (t0) readingwas taken on the day of compound addition. To develop the plates, 25 μLof Cell Titer Glo (Promega #G7572) was added to each well and incubatedat room temperature for 12 minutes. Luminescence was measured on theTecan Safire2.

Data were analyzed using Assay Client software. Dose response curveswere generated based on percent of growth compared to t0. Growth IC50values were calculated using a 4 parameter logistic model.

Compound A is an EZH2 inhibitor.

Example 10: Aberrantly Elevated H3K27Me3 Levels in Y641 Wild-TypeLymphoma Cell Line

A variety of activating and inactivating mutations of EZH2 have beendescribed in primary tumors derived from GCB DLBCL, FL, andmyelodysplastic syndrome (MDS) (1-3, 24-26). The end result of thesemutations is increased or decreased methylation of H3K27 (14, 15, 24,26). In order to characterize alterations of H3K27me3 in human cancercell lines, we quantified global H3K27me3 and total histone H3 levels byELISA in 111 cell lines from 7 unique tumor types (FIG. 1A). Each of thetumor types examined exhibited a range of H3K27me3 levels with severallymphoma cell lines possessing H3K27me3 levels 2-3 fold higher than thehighest non-lymphoma cell lines. Further analysis of protein lysatesfrom several of these cell lines revealed an apparent imbalance betweenthe methylation states of H3K27 (FIG. 1B). Overall, H3K27me3 levels wereincreased at the expense of H3K27me1 which were reduced relative tolymphoma cell lines with lower global H3K27me3 levels.

Example 11: Mutation of the A677 Residue of EZH2 to Glycine in aLymphoma Cell Line with Aberrantly Elevated H3K27Me3

Based upon recent findings demonstrating a hyper-trimethylationphenotype for H3K27 in lymphoma cells harboring mutation of the Y641residue of EHZ2 to either phenylalanine (F), asparagine (N), serine (S),or histidine (H), we hypothesized that the lymphoma cell lines with thehighest levels of H3K27me3 may have activating mutations at Y641. Sangersequencing of all cell lines for the Y641 codon revealed activatingmutations for 6 of the top 7 lymphomas when ranked by global H3K27me3levels.

The one cell line with high H3K27me3 levels that was not mutated at Y641was Pfeiffer. The Pfeiffer cell line was established in 1992 from thepleural effusion of a patient in the leukemic phase of DLBCL (27).Sanger sequencing of genomic DNA for all EZH2 coding exons revealed aheterozygous C to G mutation at nucleotide 2045 leading to anon-synonymous mutation of the A677 residue to a glycine (A677G) (FIG.2A). Sequence analysis of cDNA revealed that both wild-type and mutantalleles are expressed (data not shown). This residue falls within thecatalytic SET domain of EZH2 and is located 2 exons downstream of theY641 residue in exon 18 (FIG. 2B). This residue is highly conservedacross multiple species and multiple histone methyltransferases (FIG.2C) indicating that it may play an essential role in the function ofEZH2.

Example 12: Occurrence of the A677G EZH2 Mutation in Primary LymphomaSamples

To establish whether the mutation identified in the Pfeiffer cell lineoccurs in primary human lymphomas, this residue was sequenced in a panelof 41 lymphoma specimens. This panel consisted of 30 DLBCLs, 6 FL, 1extra nodal marginal zone B-cell lymphoma of mucosa associated lymphoidtissue (MALT), 1 mantle cell lymphoma (MCL), 1 splenic marginal zonelymphoma (SMZL), and 2 Waldenström's macroglobulinemia orlymphoplasmacytic lymphomas (WM) (Table 2). In addition to 4 occurrencesof Y641 mutation, non-synonymous missense mutations were observed forP488 and A677. The A677 mutation was heterozygous and occurred in astage IIE DLBCL obtained from a 74 year old female (FIG. 2A; Table 2).This mutation was expressed as both alleles were detected in cDNA (datanot shown). These data demonstrate that the A677G mutation occurs inprimary human lymphoma and is not simply an artifact of cell culture.

Example 13: The EZH2 A677G Mutation Confers Biochemical ActivityIndependent of H3K27 Methylation State

EZH2 Y641 mutations affect substrate specificity resulting in apreference for H3K27me2 over the unmodified and mono-methylated forms(FIG. 2B) (3, 14). To further investigate the effect of the A677Gmutation on substrate preference, we characterized the steady statekinetics of wild-type and mutant EZH2 complexes using peptide substratescontaining H3K27me0, H3K27me1, or H3K27me2. When comparing turnover withthe three peptide substrates (as either k_(cat) or k_(cat)/K_(m)), wildtype EZH2 loses activity when progressively more methyl groups areincorporated into H3K27 (i.e. H3K27me0>H3K27me1>H3K27me2) (FIG. 3, Table1). In contrast, all Y641 mutant enzymes that were evaluated displayedthe opposite trend with the H3K27me2 peptide being utilized mostefficiently (i.e. H3K27me2>H3K27me1>H3K27me0) (FIG. 3, Table 1). TheA677G EZH2 complex, on the other hand, displayed a profile differentfrom both wild-type and Y641 mutants. A677G EZH2 utilized all threesubstrates with nearly equal efficiency and at a rate comparable withthe best turnover found with the wild type enzyme (k_(cat)=0.64s⁻¹ vs0.62 s⁻¹, respectively) (FIG. 3, Table 1). When nucleosomes purifiedfrom HeLa cells were evaluated, the activity of A677G EZH2 was higherthan wild type EZH2 and the Y641 mutants (k_(cat)=0.17 s⁻¹ vs 0.10 s⁻¹for wild-type and 0.11 s⁻¹ for Y641S). This observation is likely theresult of the ability of the A677G EZH2 complex to act upon a greaterproportion of the histones which are heterogeneously modified at H3K27.

Example 14: Expression of A677G EZH2 is Sufficient to DriveHyper-Trimethylation of H3K27

To explore the effect of the A677G and Y641 EZH2 mutants on histonemethylation levels, wild-type and mutant versions of EZH2 weretransiently expressed in cells prior to evaluation of H3K27me1 andH3K27me3 levels. MCF-7 cells were selected for this analysis as theyexhibit relatively low levels of H3K27me3 (26% of Pfeiffer H3K27me3levels; FIG. 1A) and are easily transfected. Indeed, exogenousexpression of either A677G or Y641 mutant EZH2 was capable of inducing a2-3-fold increase in H3K27me3 bringing the global level of this mark toa level similar to that observed in Pfeiffer cells (FIG. 4). These datademonstrate that expression of the A677G is sufficient to induce aglobal hyper-trimethylation of the H3K27 residue.

Example 15: Structural Rationale for the Substrate Preference andProduct Specificity of A677G Mutant EZH2

In the absence of an EZH2 crystal structure, a homology model wasconstructed based on the protein sequence of wild-type EZH2 and acrystal structure of GLP/EHMT1 bound to a histone H3 peptide substratecontaining H3K9me2 (FIG. 5A). This model is consistent with thebiochemical data demonstrating that wild-type EZH2 primarily catalyzesmono- and di-methylation, but not tri-methylation, of H3K27. Similar toother methyltransferases such as Set7/9, the highly conserved EZH2 Y641residue appears to orient the unmethylated and mono-methylated forms ofH3K27 for optimal methyl transfer through hydrogen bonds with the lysinee-amine group (28, 29). In addition, the presence of a conservedphenylalanine residue (F724) instead of a tyrosine at the Phe/Tyr switchposition results in the loss of a hydrogen bond to a structurallyconserved water molecule (not depicted in FIG. 5) which has been shownto further orient the un-methylated lysine (28). Since the conservedwater lacks the additional hydrogen bond, it is presumably more weaklybound and therefore more easily displaced by the mono-methylated lysinewhen it rotates into position for the second methyl transfer. Thus, thismodel supports EZH2 functioning efficiently as a mono- anddi-methyltransferase. However, there is very little room for adi-methylated lysine to rotate into position to accept a third methylgroup with only 3.3 Å between the hydroxyl oxygen of Y641 and thee-amine group of H3K27me2. Thus, the Y641 residue of EZH2 has a dualpurpose participating in the orientation of unmethylated andmono-methylated lysine while at the same time sterically restrictingtri-methylation.

This model predicts that mutation of Y641 to a smaller residue such asasparagine (Y641N) results in both loss of a critical hydrogen bond andgeneration of a larger lysine tunnel (FIG. 5B). The larger lysine tunneland loss of proper hydrogen bonding presumably hinder stabilization ofan unmodified or mono-methylated lysine which is highly flexible. On theother hand, replacing Y641 with smaller residues, such as asparagine,generates a larger tunnel (H3K27me2 methyl carbon to N641 side chaindistance=3.8 Å) which permits rotation of the di-methylated lysine intoposition for a third methyl transfer. Presumably the two methyl groupsof H3Kme2 also make advantageous hydrophobic interactions in the lysinetunnel that help to further orient the di-methylated lysine. Thisinterpretation is consistent with data presented by us and othersdemonstrating that Y641 mutants do not efficiently utilize anunmethylated lysine, yet have acquired robust activity withdi-methylated substrates.

Interestingly, while the structural model for wild-type EZH2 suggeststhat the highly conserved A677 residue does not interact directly witheither SAM or H3K27, it is in close proximity to the hydroxyl oxygen ofY641 (3.3 Å) (FIG. 5A). It is therefore predicted that mutating A677 toa smaller glycine residue permits Y641 to adopt an alternativeconformation which generates additional space within the lysine tunnelallowing the di-methylated substrate to rotate into an orientationsuitable for methyl transfer (FIG. 5C). This alternative Y641conformation has sufficient space (3.1 Å) between the hydroxyl oxygen ofY641 and the closest methyl carbon of H3K27me2 even when the lysine tailis rotated into position for a third methyl transfer. Thus, this modelsuggests that the retention of the Y641 residue combined with thealternative Y641 orientation contributes to the efficient methylation ofunmodified, mono-, and di-methylated substrate.

Example 16: Cell Line Sensitivity to EZH2 Inhibitors in ProliferationAssay (Assay 1)

To evaluate the effects of EZH2 mutations and H3K27me3 levels on thesensitivity to EZH2 inhibition, 38 lymphoma cell lines were evaluated ina 6 day proliferation assay (assay 1) using an EZH2 inhibitor indicatedas compound A. As shown in Table 5 and FIG. 6, cell lines which possessa mutation in EZH2 and have high H3K27me3 (≧100% of Pfeiffer) aresensitive to EZH2 inhibition. EZH2 mutation status alone does notpredict sensitivity to EZH2 inhibition under these conditions as thereare number cell lines (e.g. SUDL-4, DB, OC1-LY-19 & SKM1) with an EZH2mutation are resistant or weakly sensitive to EZH2 inhibition. Inaddition, further optimization of the EZH2 inhibition assay

Example 17: Discussion of EZH2 Mutations, Cancer, and Treatment withEZH2 Inhibitors

Elevated levels of EZH2 is a hallmark of many cancers and appears to berequired for the proliferation of some lymphoma cells as knockdown ofEZH2 in the SU-DHL-4 DLBCL cell line results in growth arrest at theG1/S transition (30). Recent studies have identified mutations in EZH2at Y641 in lymphoma which result in H3K27 hyper-trimethylation (1, 3,14, 15). Herein, we have reported the identification andcharacterization of a novel EZH2 mutation capable of increasing globalH3K27me3 in human lymphoma cells and demonstrate that lymphoma celllines with elevated levels of H3K27me3 and harboring either a Y641 orA677 mutation in EZH2 are sensitive to inhibition of EZH2.

While the EZH2 Y641 mutation occurs in 22% of GCB DLBCL and 7% of FL(3), our study indicates that mutation of the EZH2 A677 to glycine is afairly rare event. We observed this mutation in 1 of 50 lymphoma celllines and 1 of 41 primary lymphoma samples. In addition, Morin et alrecently observed a single case of DLBCL (63 yo female stage 1AE) withthe A677G mutation among 127 samples that were assessed by RNA-seq,exome-seq, and/or genome-seq (1). Thus, while a more extensive studywith focused genotyping of the A677 codon will be required to establishthe true incidence of this alteration, these initial data suggest thatthe frequency of this mutation is likely below 2-3%.

Considering that the end result (i.e. increased H3K27me3) of these twomutations may be quite similar, it is at first somewhat surprising thatthese mutations occur at such different rates. However, this discrepancymight be explained in large part by the spectrum of possible activatingmutations at each site. To date, mutation of Y641 to any of 5 differentresidues (F, N, S, C, or H) has been reported to increase activity withan H3K27me2 substrate ((1, 3, 14, 15) and this study). This increasedactivity has been attributed to the exchange of Y641 for smallerresidues which permit the larger H3K27me2 substrate to rotate into aposition for methyl transfer. The A677G mutation appears to similarlyincrease the dimensions of the lysine tunnel through exchange of alaninefor a smaller amino acid; however, since alanine is already the secondsmallest amino acid, it may only be exchanged for a glycine. At thenucleotide level, only 1 of 9 single nucleotide mutations within theA677 codon will achieve a glycine residue, whereas 5 of 9 achieve anactivating mutation at Y641. Thus, the apparently low incidence of theEZH2 A677G mutation may simply be due to the extremely limited number ofpossible alterations for this particular site.

The fact that the SET domain is highly conserved across orthologous andhomologous methyltransferases readily permits translation of findingsfrom one methyltransferase to another. For example, the effect ofchanges at the Y641 residue of EZH2 are predictable based on mutationalanalyses of other SET domain methyltransferases whose biochemicalproperties have been more extensively studied. The human SET7/9methyltransferase normally mono-methylates H3K4, however, when theSET7/9 Y245 residue (the equivalent of EZH2 Y641) is mutated to alanine,the mutant can no longer modify a H3K4me0 substrate, but instead gainsthe ability to di- and tri-methylate an H3K4me1 peptide substrate (31).An additional study with the H3K9 methyltransferase G9a demonstratedthat mutation of Y1067 to phenylalanine converts the enzyme from a mono-and di-methyltransferase to a tri-methyltransferase (32).

Example 18: EZH2 A677 Mutant, e.g. EZH2 A677G is a Novel Biomarker forUse in Therapy

To the best of our knowledge, this study is the first report to examinethe biochemical and cellular activity of the A677 residue of EZH2 whichis conserved across multiple PKMTases. Interestingly, however, thestructurally related SET domain containing DIM-5 from N. crassa has aglycine at the equivalent position (FIG. 2C) and has been reported toperform all three methylation events on its H3K9 substrate (33, 34).Thus, it appears that the alanine at residue 677 of EZH2, and likelyequivalent residues in other SET domain methyltransferase, plays animportant role in the regulation of substrate specificity without beingin direct contact with the substrate. This interplay between Y641 andA677 in EZH2 highlights just one of the many important mechanisms thathave likely evolved to regulate the substrate and product specificitiesof lysine methyltransferases.

Example 19: Overview of EZH2 Inhibitor Compound B Treatment in EZH2Mutants In Vitro and In Vivo

In eukaryotes, epigenetic post-translational modification of histones iscritical for regulation of chromatin structure and gene expression. EZH2is the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) andis responsible for repressing gene expression through methylation ofhistone H3 on lysine 27 (H3K27). EZH2 over-expression is implicated intumorigenesis and correlates with poor prognosis in multiple tumor types(21, 36-39). Additionally, somatic heterozygous mutations of Y641 andA677 residues within the catalytic SET domain of EZH2 occur in diffuselarge B-cell lymphoma (DLBCL) and follicular lymphoma (FL) (1, 3,40-42). The Y641 residue is the most frequently mutated residue, with upto 22% of GCB (Germinal Cell B-cell) DLBCL and FL harboring mutations atthis site. These lymphomas exhibit increased H3K27 tri-methylation(H3K27me3) due to altered substrate preferences of the mutant enzymes(14, 15, 41, 43). However, it is unknown whether specific, directinhibition of EZH2 methyltransferase activity will be effective intreating EZH2 mutant lymphomas. Herein, we demonstrate that GSK126, apotent, highly-selective, S-adenosyl-methionine (SAM)-competitive, smallmolecule inhibitor of EZH2 methyltransferase activity, decreases globalH3K27me3 levels and reactivates silenced PRC2 target genes. GSK126effectively inhibits the proliferation of EZH2 mutant DLBCL cell linesand dramatically inhibits the growth of EZH2 mutant DLBCL xenografts inmice. Together, these data demonstrate that pharmacological inhibitionof EZH2 activity may provide a promising treatment for EZH2 mutantlymphoma.

Compound B (GSK126) is an EZH2 inhibitor. Compound B has the formula:

Example 20 High Throughput Screen to Identify Compound B (GSK126) as aPotent Inhibitor of EZH2 Methyltransferase Activity

To identify inhibitors of EZH2 methyltransferase activity, ahigh-throughput biochemical screen with a 5-member PRC2 protein complexwas performed (44). This work identified a small molecule EZH2 inhibitorwith a K_(i) ^(app)=700 nM. Extensive optimization of this compoundthrough medicinal chemistry generated GSK126 (FIG. 7A). GSK126 potentlyinhibits both WT and mutant EZH2 methyltransferase activity with similarpotencies (K_(i) ^(app)=0.5-3 nM) independent of substrate utilized, andis competitive with SAM and non-competitive with peptide substrates(FIG. 7B, data not shown). GSK126 is highly selective against othermethyltransferases and protein classes (Data not shown). In particular,GSK126 is >1,000-fold selective for EZH2 versus 20 other humanmethyltransferases, including both SET domain and non-SET domaincontaining methyltransferases (45). Even EZH1, which is 96% identical toEZH2 within the SET domain, and 76% identical overall, isinhibited >150-fold less potently (K_(i) ^(app)=89 nM). Utilizing anEZH2 homology model (41), combined with enzyme mechanism-of-action andinhibitor structure-activity relationship data, in silico dockingrevealed the SAM binding pocket as the most plausible docking site forGSK126. Here it is predicted to make extensive contacts with thepost-SET domain which forms one side of the SAM binding pocket (FIG.8a-d ) Interestingly, within 10 Å of the predicted GSK126 binding site 4of the 6 residue differences between EZH2 and EZH1 lie within thepost-SET domain and these may contribute to the loss of potency forEZH1.

Example 21 Compound B (GSK126) Induces Loss of H3K27Me3

The altered substrate preferences of EZH2 mutants lead to an imbalancein cellular H3K27 methylation states (FIG. 9a ) (14, 41). Nonetheless,GSK126 induced loss of H3K27me3 in both EZH2 WT and mutant DLBCL celllines with IC₅₀ values ranging from 10-252 nM independent of EZH2mutation status (T-test, p=0.27) (FIG. 7c ). Further analysesdemonstrated that inhibition of H3K27me3 began before 24 hours andpotency was maximal after 2 days (FIG. 9b ). GSK126 most potentlyinhibited H3K27me3, followed by H3K27me2, and H3K27me1 was only weaklyreduced at the highest inhibitor concentration (FIG. 7, and FIG. 9c ).Total histone H3 and PRC2 components were not affected by GSK126 (FIGS.9c and 10), thus reduction of H3K27 methylation is due to directinhibition of EZH2 methyltransferase activity and not degradation ofhistone H3 or PRC2. This is in contrast to 3-deazaneplanocin A (DZNep),an inhibitor of S-adenosyl-L-homocysteine (SAH) hydrolase that promotesdegradation of the PRC2 complex and indirectly inhibits EZH2 througheffects on intracellular SAH concentrations (46).

Example 22: Compound B Inhibits Growth of B Cell Lymphoma Cells

We evaluated the effect of Compound B (GSK126) on cell proliferation ina panel of B-cell lymphoma cell lines, using an improved proliferationassay as compared to hat used for Compound A (Assay 1 was used forCompound A treatment, above.). Thus, Example 22 and Table 6 betterreflect the inhibition of HZH2 inhibitors on cell lines, e.g. thelymphoma cell lines. DLBCL cell lines were the most sensitive to EZH2inhibition (FIG. 11a ). Six of the seven most sensitive DLBCL cell linesharbored Y641N, Y641F, or A677G EZH2 mutations (growth IC₅₀=28-861 nM)(FIG. 11a , Table 6, and FIG. 12). The exception was HT which is WT forEZH2 (growth IC₅₀=516 nM). Interestingly, HT harbors a mutation in UTX(R1111C), a H3K27 demethylase frequently inactivated in multiple tumortypes (19). Only 2 of the 11 remaining DLBCL cell lines harbored EZH2mutations suggesting that, in most cases, DLBCL cell lines with mutantEZH2 are dependent on EZH2 activity for cell growth; however, in somesituations co-occurring alterations may override the cell's dependenceon EZH2 activity making it less sensitive to EZH2 inhibition. Among EZH2mutant cell lines, sensitivity to GSK126 is independent of BCL2translocation or p53 mutation, common alterations found within DLBCL(Table 6). There was a modest correlation between inhibition of H3K27me3and cell growth (Pearson, r=0.62), but there was no correlation betweensensitivity to GSK126 and EZH2 protein levels (FIG. 13a-c ).Interestingly, two of the most sensitive DLBCL cell lines, WSU-DLCL2 andKARPAS-422, are derived from patients with refractory disease (47, 48)suggesting that DLBCL cells that are resistant to standard-of-care maybe sensitive to EZH2 inhibition. Burkitt (BL) and Hodgkin's (HL)lymphoma cell lines were generally less sensitive to EZH2 inhibition(growth IC₅₀>1.3 μM) with the exception of Jiyoye (growth IC₅₀=0.23 μM),a BL cell line with WT EZH2. Evaluation of GSK126 in additional lymphomacell lines and extensive genomic and epigenomic characterization will berequired to fully elucidate the determinants of sensitivity amonglymphoma subtypes.

Both cytostatic and cytotoxic responses were observed among the mostsensitive cell lines (Table 6); therefore, the timing of GSK126-inducedeffects on proliferation and cell death was examined in detail in two ofthe most sensitive cell lines. In Pfeiffer, potent inhibition of cellproliferation was observed after 2 days (FIG. 11b ) and net decreases incell number were evident after 3 days (FIG. 11c ). This cell deathappears to be driven by caspase-mediated apoptosis as indicated by theincrease in the sub-G₁ population (FIG. 11d ) and dose-dependentinduction of caspase activity (FIG. 11e ). The response in KARPAS-422was slower with 6-7 days required for maximal potency (FIG. 11b ).Furthermore, a primarily cytostatic effect was observed in KARPAS-422 asdemonstrated by CTG values remaining above day 0 levels, a G₁ arrest(43% and 77% of cells in G₁ with DMSO and 500 nM GSK126, respectively)with little sub-G₁ content, and minimal caspase activity with <1 μMGSK126 (FIG. 11f-h ). Consistent with these observations, shRNA-mediatedknockdown of EZH2 led to profound cytotoxic and apoptotic responses inPfeiffer, and decreased cell proliferation, and no caspase activation inKARPAS-422 demonstrating that the phenotypic effects observed withGSK126 are due to inhibition of EZH2 (FIG. 14a-c ).

Example 23 the Effect of Compound B on Gene Expression

Since EZH2 is associated with transcriptional repression, we evaluatedthe effect of GSK126 on gene expression in DLBCL cell lines with a rangeof sensitivity to GSK126. Robust transcriptional activation was noted inthe most sensitive cell lines (FIG. 15a , FIG. 16a , and data notshown). Not surprising, considering the repressive nature of H3K27me2/3,the majority of transcriptional changes involved up-regulation. The highdegree of similarity between gene expression changes observed withGSK126 treatment and EZH2 knockdown in KARPAS-422 and Pfeiffer cellssuggests that these transcriptional changes are due to loss of EZH2activity and not off-target effects (data not shown Additionally,analysis of ChIP-seq data for the 3 most responsive cell lines revealedthat prior to treatment up-regulated genes exhibited broad enrichment ofH3K27me3 suggesting these genes are EZH2 targets marked by H3K27me3(FIG. 15b and FIG. 17a-c ).

In contrast to the response observed in the sensitive cell lines,minimal transcriptional changes occurred with GSK126 treatment inToledo, a cell line with WT EZH2 whose growth is not affected by EZH2inhibition (FIG. 15a , FIG. 16b ). Even at 2 μM GSK126, very fewtranscriptional changes were observed in Toledo (23 up-regulated and 10down-regulated probe sets), despite a near complete loss of H3K27me3 atthis dose and time (FIG. 9c and data not shown). Likewise, qRT-PCRperformed for two H3K27me3-enriched genes revealed dose-responsiveincreases in gene expression with as little as 25 nM GSK126 in Pfeifferand KARPAS-422, but no transcriptional changes in Toledo with up to 1 μMGSK126 (FIG. 15c , data not shown). Interestingly, even in the mostsensitive WT EZH2 cell line, HT, the transcriptional response was lesspronounced when compared to EZH2 mutant DLBCL cell lines with similarsensitivity (FIG. 15a ). Relaxing the transcriptional fold-changecriteria from 2.0 to 1.5 revealed additional modest transcriptionalchanges in HT cells (FIG. 16b ). This muted transcriptional response inEZH2 WT and less sensitive mutant cell lines suggests that othercompensatory mechanisms (such as H3K9, H4K20, or DNA methylation) mayexist in these cell lines to dampen the transcriptional response.

Among the EZH2 mutant cell lines, global H3K27me3 levels werestatistically higher in transcriptionally-responsive lines (T-test,p=0.019) suggesting that EZH2 mutation status together with globalH3K27me3 levels may be a better predictive biomarker than mutationstatus alone (FIG. 16c ). While the five most sensitive EZH2 mutant celllines exhibited a preponderance of up-regulated gene expression changes(69-95%), little overlap was observed among the differentially-regulatedprobe sets using 2-fold or 1.5-fold significance criteria (FIG. 15d andFIG. 16d ). Only 35 up-regulated probe sets were common to at least 4 ofthese 5 mutant cell lines (Table 7). Examination of these commonlyup-regulated probe sets revealed that many are enriched for H3K27me3(32/35) (Table 7 and data not shown). Additionally, many of these probesets are induced, albeit weakly, in the other cell lines suggesting thatadditional time or chromatin factors may be required for complete geneactivation in these settings (FIG. 15e and Table 7). Lastly, while nosingle pathway or process was significantly enriched among the limitedset of genes commonly up-regulated, gene ontology enrichment analysis ofregulated gene sets in each cell line individually revealed severalcommon processes including cell cycle regulation, cell death, andregulation of biological/cellular processes (FIG. 18 and data notshown). These data demonstrate that the global loss of H3K27me3following inhibition of EZH2 with GSK126 is associated withtranscriptional activation of EZH2 target genes that correlates wellwith sensitivity and that mutant EZH2 de-regulates H3K27me3 in a global,rather than targeted, manner. The significant variation between theup-regulated gene sets of sensitive cell lines is a surprisingobservation that likely highlights the complexity and uniqueness of theepigenome in each cell line, and the diversity of selective pressuresduring the development of individual lymphomas.

Example 24 Compound B Inhibits Tumor Growth In Vivo

Based upon the potent effects in cell culture, we evaluated GSK126 inmice using subcutaneous xenografts of KARPAS-422 and Pfeiffer. Following10 days of once-daily (QD) dosing of GSK126, global H3K27me3 decreasedand gene expression increased in a dose-dependent fashion consistentwith observations from cell culture (FIG. 19a,b ). Although GSK126 wasinitially cleared rapidly from the blood, there was an extended terminalphase where drug elimination from blood and tumor was slower (FIG. 20a,b). With daily 50 mg/kg dosing, complete tumor growth inhibition wasobserved in both KARPAS-422 and Pfeiffer models (FIG. 19c , and FIG. 21a). When higher dosing regimens were examined with KARPAS-422 xenografts,marked tumor regression was observed (FIG. 19c ). Upon cessation ofdosing, tumors in the 50 mg/kg QD group exhibited tumor stasis whilecomplete tumor eradication was observed in the 150 mg/kg QD and 300mg/kg twice/week groups. Tumor growth inhibition also correlated withstatistically significant increased survival of mice bearing the moreaggressive KARPAS-422 tumors, where spontaneous deaths occurred invehicle-treated animals (FIG. 19d ). Based upon these strikingobservations, intermittent dosing regimens with lower doses of GSK126given weekly or with a 1 week drug holiday were examined in KARPAS-422tumor xenografts with large tumors (FIG. 21b ). All schedulesdemonstrated tumor growth inhibition (91-100%, T-test, p values=0.0008to 0.0024). These results indicate that the response to GSK126 isdurable and that intermittent dosing schedules may be effective in aclinical setting even in advanced tumors.

GSK126 was well tolerated at the doses and schedules examined asmeasured by little to no decrease in body weight, normal grooming andbehavior, and vastly improved survival in mice carrying KARPAS-422xenografts (FIG. 22a-c and FIG. 19d ). Given the role of EZH2 in normalhematopoiesis and the identification of EZH2 loss-of-function mutationsin myeloid malignancies (49-52), we investigated the effects of GSK126treatment on peripheral blood of immunocompetent mice. Complete bloodcount analysis revealed no significant changes in any blood cell typesat doses and times where efficacy was observed in tumor xenografts (FIG.22d ).

Over the past decade, the development of targeted agents thatspecifically inhibit oncoproteins with activating somatic alterationshas provided profound clinical benefit for cancer patients (53, 54). Thedata herein provide compelling evidence that inhibition of EZH2methyltransferase activity may be a viable strategy for the treatment ofDLBCL and non-indolent FL harboring activating mutations in EZH2. GSK126also provides a means to evaluate whether EZH2 activity is required forthe survival of tumors where EZH2 over-expression has been linked topoor prognosis (36-39), and tumors harboring loss-of-function mutationsin UTX (19, 50, 55). While we do not expect GSK126 to be effective intreating myeloid malignancies bearing loss-of-function mutations in EZH2(50-52), GSK126 should be an important tool to assess the role of EZH2in normal myeloid development and to understand the oncogenic role ofEZH2 in myeloproliferative neoplasms. Lastly, the identification of aselective EZH2 inhibitor which does not lead to degradation of the PRC2complex provides a useful tool to understand the role of EZH2methyltransferase activity versus its scaffolding role in development,tumorigenesis, and tumor progression that could not be elucidatedthrough conventional genetic manipulation studies.

Example 25: Methods Summary for Examples Disclosing GSK126 (Compound B)

Biochemical assays utilized the 5-member PRC2 complex (human Flag-EZH2,EED, SUZ12, AEBP2, RbAp48) containing either WT or mutant EZH2, [³H-]SAMand the indicated peptide substrate; reactions were incubated for 30minutes. Global histone modification levels were determined by ELISA orwestern blot methods using antibodies specific for total histone H3,H3K27me1, H3K27me2, or H3K27me3. Cell proliferation and Caspase-3/7activity were assessed using CellTiter-Glo and Caspase-Glo 3/7(Promega), respectively. Gene expression profiling was conducted usingAffymetrix Human Genome U133 Plus 2.0 microarrays.Differentially-expressed probe sets were determined by fitting the datato a linear model using the limma statistical package(http://www.bioconductor.org) and carrying out pair-wise contrasts oftreated versus control. Significant probe sets were filtered fordetection (log₂ signal threshold of 8), an average fold-change >2 or<−2, or >1.5 or <−1.5, where indicated, with p-values adjusted formultiple testing correction by FDR (Benjamini Hochberg)<0.1. H3K27me3ChIP reads were aligned using Bowtie (56). H3K27me3 enrichment peakswere identified using SICER (57) with optimized parameters. A customPERL script was utilized to quantify the average basal H3K27me3 ChIP-seqtag density across gene sets. All in vivo studies were conducted afterreview by the Institutional Animal Care and Use Committee at GSK and inaccordance with the GSK Policy on the Care, Welfare and Treatment ofLaboratory Animals. GSK126 and vehicle were administered to miceintraperitoneally. Two-tailed t-tests were conducted assuming twosamples of equal variance.

Determination of K?^(a)PP Values for GSK126 Inhibition of WT and MutantEZH2.

The 5 member PRC2 complex (Flag-EZH2, EED, SUZ12, AEBP2, RbAp48)containing either WT or mutant (A677G, Y641N, Y641C, Y641H, Y641S, orY641F) EZH2 was prepared as previously described (41). GSK126 wasdissolved in DMSO and tested at concentrations of 0.6 nM to 300 nM witha final DMSO concentration of 2.5%. In contrast to wild-type EZH2 whichprefers H3K27me0 as a substrate in vitro, EZH2 Y641 mutants preferH3K27me2 and have little activity with H3K27me0 or H3K27me1. The A677Gmutant is distinct from both the wild-type and Y641 mutant forms of EZH2in that it efficiently methylates H3K27me0, H3K27me1, and H3K27me2;therefore, histone H3 peptides (residues 21-44; 10 μM final) with eitherK27me0 (WT, A677G EZH2), K27me1 (A677G EZH2), or K27me2 (A677G, Y641N,Y641C, Y641H, Y641S, and Y641F EZH2) were used as methyltransferasesubstrates. GSK126 was added to plates followed by addition of 6 nM EZH2complex and peptide. As the potency of GSK126 is at or near the tightbinding limit of an assay run at [SAM]=K_(m), we used a method whereIC₅₀ values were measured at a high concentration of the competitivesubstrate SAM relative to its K_(m) (7.5 μM SAM where the SAM K_(m) is0.3 μM). Under these conditions, the contribution from the enzymeconcentration becomes relatively small (see EQ 1) and accurate estimatesof K_(i) can be calculated (58). Reactions were initiated with [³H-]SAM,incubated for 30 minutes, quenched with the addition of 500-fold excessunlabeled SAM, and the methylated product peptide was captured onphosphocellulose filters according to the vendor supplied protocol forMSPH Multiscreen plates (EMD Millipore, Billerica, Mass., USA). Plateswere read on a TopCount after adding 20 μL of Microscint-20 cocktail(both from PerkinElmer, Waltham, Mass. USA). Apparent K_(i)values+/−s.d. were calculated using the Cheng-Prusoff relationship (59)for a competitive inhibitor (n=2).

IC₅₀ =K _(i)*(1+[S]/K _(m))+[E]/2.  EQ1:

Mechanism of GSKJ26 Inhibition of EZH2.

IC₅₀ values were determined for GSK126 inhibition of EZH2 at several SAMconcentrations ranging from 0.1 μM to 15 μM and then separately atseveral peptide concentrations ranging from 16 μM to 60 μM using theassay conditions described above. The resulting IC₅₀ values were plottedagainst the [SAM]/K_(m) ratio or the [peptide]/K_(m) ratio respectively.

Cell Culture and Immunoblotting.

Cell lines were obtained from the American Type Culture Collection orthe Deutsche Sammlung von Mikroorganismen und Zellbulturen andmaintained in the recommended cell culture media at 37° C. in 5% CO₂.Cells were lysed with radioimmunoprecipitation (RIPA) buffer(ThermoScientific) and western blot analysis was conducted as previouslydescribed (41). Antibodies were obtained as previously described (41) orfrom Cell Signaling Technology (SUZ12, 3737), or Santa CruzBiotechnology (EED, sc-28701).

H3K27 Methylation Status and PRC2 Components Following GSK126 Treatment.

Cells (2×10⁵/well) were seeded into six-well tissue culture plates inthe appropriate cell culture media 24 hours before treatment. Cells werethen exposed to 0.1% DMSO or varying concentrations of GSK126 (range=25nM-2 μM) for 24, 72, or 144 hours.

ELISA-Based Quantitation of Total Histone H3 and H3K27Me3 Levels.

Following tissue homogenization, tumor tissue lysates were preparedusing the Epigentek Histone Extraction kit (OP-0006). Alternatively,cells were seeded at 2,000 cells/well in a 96-well plate and weretreated with a 10-point 3-fold dilution series of GSK126 (dose range=2nM-38 μM) for 48 hours. Cells were lysed with 0.2N HCl for 30 minutes toextract histones, the acid-insoluble portion was pelleted bycentrifugation, and the supernatant was neutralized with neutralizationbuffer (1M Na₂HPO₄, pH 12.5; ActiveMotif) containing protease inhibitors(Roche). Lysates were added to Maxisorp ELISA plates (Nunc) in duplicateon each of two plates plus blocking buffer (1% BSA). Plates wereincubated for 1 hour, washed 4 times with imidazole buffered salinecontaining Tween-20 (Kirkegaard & Perry Laboratories), incubated withprimary antibodies for H3K27me3 or total H3, washed, incubated withHRP-linked secondary anti-rabbit IgG antibody, and washed again.Luminata Forte substrate (Millipore) was added 5 minutes beforechemiluminescence was quantified with an EnVision multi-label platereader (PerkinElmer). H3K27me3 levels were normalized to total H3 valuesand IC₅₀ values were determined using a 4-parameter curve fit.

Cell Proliferation Assay.

The optimal cell seeding was determined empirically for all cell linesby examining the growth of a wide range of seeding densities in a384-well format to identify conditions that permitted proliferation for6 days. Cells were then plated at the optimal seeding density 24 hoursprior to treatment (in duplicate) with a 20-point 2-fold dilution seriesof GSK126 or 0.15% DMSO. Plates were incubated for 6 days at 37° C. in5% CO₂. Cells were then lysed with CellTiter-Glo (Promega) andchemiluminescent signal was detected with a TECAN Safire2 microplatereader. In addition, an untreated plate of cells was harvested at thetime of compound addition (T₀) to quantify the starting number of cells.CTG values obtained after the 6 day treatment were expressed as apercent of the T₀ value and plotted against compound concentration. Datawere fit with a 4-parameter equation to generate a concentrationresponse curve and the concentration of GSK126 required to inhibit 50%of growth (gIC₅₀) was determined.

Caspase 3/7 Assay.

For detection of caspase-3/7 activity, cells were cultured in 96-wellplates, treated with a 10-point 3-fold dilution series of GSK126 (range0.03 nM to 5 μM) and evaluated using Caspase-Glo 3/7 (Promega) as perthe manufacturer's instructions. Values were normalized to CellTiter Glo(Promega) levels at each time point and expressed as a percentage ofvehicle treated control. Data represent an average of n=4.

Cell Cycle Analysis.

Cell cycle phase distribution was examined by flow cytometry.Twenty-four hours after seeding cells in a 6-well culture plate, cellswere treated with GSK126 or 0.1% DMSO (vehicle) for 3 days. Cells werewashed with PBS, pelleted in BD buffer solution, flash frozen, andstored at −80° C. CycleTest™ PLUS DNA reagent kit (Becton Dickinson,340242) was used according to the manufacturer's instructions to prepareand stain nuclei with propidium iodide. Samples were evaluated using aFACSCalibur flow cytometer (Becton Dickinson) and data were analyzedusing FlowJo software (Tree Star).

Gene Expression Profiling.

Cells (2×10⁵/well) were seeded into six-well tissue culture plates inthe appropriate cell culture media 24 hours before treatment. Duplicatewells were then exposed to 0.1% DMSO, 500 nM, or 2 μM GSK126 for 72hours. Cells were collected into Trizol reagent (Invitrogen) and totalRNA was isolated via phenol:chloroform extraction and the RNeasy kit(Qiagen) according to the manufacturer's instructions. Total RNA waslabeled and hybridized to Affymetrix Human Genome U133 Plus 2.0oligonucleotide microarrays arrays according to the manufacturer'sinstructions (Affymetrix, Santa Clara, Calif., USA) at ExpressionAnalysis (Durham, N.C.). These data are accessible through GEO viaaccession number GSE40972. Principal component and correlation analysiswere used to confirm data reproducibility (FIG. 16).

Affymetrix Gene Chip Data Analysis.

CEL files, corresponding to individual samples, were processed by theMicro Array Suite 5.0 (MASS) algorithm(http://www.affyrmetrix.com/support/index.affx) where signal values werescaled to a target intensity of 500 and log₂ transformed. Differentiallyexpressed probe sets were determined by fitting the data to a linearmodel and carrying out pair-wise contrasts of treated versus control.Significant probe sets were filtered for detection (log₂ signalthreshold of 8), an average fold-change >2 or <−2, or >1.5 or <−1.5,where indicated, with p-values adjusted for multiple testing correctionby FDR (Benjamini Hochberg)<0.1. Statistical analyses were performedusing the limma package from Bioconductor(http://www.bioconductor.org/). Functional analyses of differentiallyexpressed probe sets were performed using DAVID(http://david.abcc.ncifcrf.gov/). Significantly over-represented GOBiological Process (BP) and Molecular Function (MF) terms (levels 3-5)were filtered for EASE p-value <0.01.

qRT-PCR.

Cells were treated for 72 hours with 0.1% DMSO or a range ofconcentrations of GSK126 (range=25 nM-1 μM) and total RNA was isolatedas described above. RNA (2.8 μg) was reverse transcribed withMultiScribe Reverse Transcriptase (Applied BioSystems) according to themanufacturer's recommendations. The resulting cDNA was diluted and usedalong with TaqMan gene expression assays (Applied Biosystems; GAPDH,Hs03929097_g1; TNFRSF21, Hs00205419_m1; TXNIP, Hs00197750_m1). TaqManGene Expression Master Mix (Applied BioSystems), and a ViiA 7 Real-TimePCR System (Applied BioSystems) according to the manufacturer'srecommendations to quantify gene expression.

ChIP-Seq.

Cells (5×10⁷) were maintained in the appropriate cell culture media for24 hours prior to fixation. Cells were fixed for 15 minutes at roomtemperature with freshly prepared formaldehyde solution (finalconcentrations 1% formaldehyde, 10 mM NaCl, 0.1 mM EDTA pH 8.0, 5 mMHEPES pH 7.9) followed by the addition of glycine to 125 mM. Fixed cellswere rinsed twice in PBS containing 0.5% Igepal CA-630 (Sigma) and cellpellets were flash frozen. ChIP assays were performed using a customassay protocol at ActiveMotif Inc. (San Diego, Calif.). H3K27me3 ChIPand input libraries were prepared for 35 nucleotide single-endsequencing on an Illumina GAIIx sequencer according to manufacturer'sinstructions. These data are accessible through GEO via accession numberGSE40970. Reads were assessed for quality (base quality <20 wereexcluded) and aligned to human reference sequence (hg19 build) using theBowtie²⁷ algorithm allowing for up to 2 mismatches. Only uniquely mappedreads were utilized for subsequent analyses.

ChIP-Seq Analysis.

The average basal H3K27me3 ChIP-seq tag count was quantified acrossgenes that were up-regulated, down-regulated, or unchanged followingtreatment with GSK126 using a custom PERL script. In addition to thegene body, a region encompassing 10 kb upstream of the transcriptioninitiation site and 10 kb downstream of the transcription terminationsite were evaluated. All genes were oriented by strand, and the variablelength of gene bodies were standardized to 10,000 bins. After averagingthe numbers of sequence tags at each base pair the values werenormalized to the total number of mapped sequence tags per ChIP. A 500base pair centered moving average was then applied to highlight largertrends and smooth out short-range fluctuations. MultiExperiment Viewer(http://www.tm4.org/mev/) was used to evaluate enrichment acrossindividual genes. Peaks of H3K27me3 enrichment were identified using thepeak calling software SICER²⁸ with the following parameters: fragmentsize: 250 bp; effective genome size fraction: 0.86; window size: 750 bp;gap size: 3; redundancy threshold: 1; FDR: 0.001. Statisticallysignificant peaks (FDR<0.001) enriched in the ChIP sample relative toits corresponding input sample were annotated for genomic location andwere assigned to genes within +/−10 kb from transcription start site(TSS) to identify target genes: upstream (−10 to 2.5 kb relative toTSS); promoter (−2.5 kb to +2.5 kb); 5′UTR; coding region; 3′UTR. Allgenes were considered in the 5′->3′ orientation. Bedtools was used formanipulation and analysis of data and IGVhttp://www.broadinstitute.org/igv was used for visualization. Annotationfiles were downloaded from UCSC. Functional analyses of differentiallyexpressed probe sets were performed using DAVID(http://david.abcc.ncifcrf.gov).

RNA Isolation from Tumor Xenografts.

Qiazol (300 μl/mg tumor) (Qiagen) was added to tumor xenograft tissue.The tumor was lysed and homogenized using the Qiagen TissueLyzer andstainless steel beads. Chloroform was added to the Qiazol lysate. TheQiazol/chloroform homogenate was then added to a Qiagen MaXtract HighDensity tube (Qiagen). The aqueous phase was transferred to a fresh tubeand mixed with an equal volume of 70% EtOH and applied to a QiagenRNeasy column (Qiagen). The remaining RNA isolation was carried outaccording to the manufacturer's protocol.

In Vivo Studies.

All studies were conducted after review by the Institutional Animal Careand Use Committee at GSK and in accordance with the GSK Policy on theCare, Welfare and Treatment of Laboratory Animals. For all in vivostudies, GSK126 or vehicle was administered intraperitoneally at a dosevolume of 0.2 mL/20 g body weight in 20% captisol adjusted to pH 4-4.5with 1 N acetic acid. Pfeiffer or KARPAS-422 cells (1×10⁷) in 100%matrigel (BD Biosciences) were implanted subcutaneously in female beigeSCID mice. Tumors were measured with calipers, block randomizedaccording to tumor size into treatment groups. For efficacy studies, 10mice were randomized in each treatment group prior to the initiation ofdosing and GSK126 treatment was initiated once the tumor volumes wereapproximately 200 mm³ in the Pfeiffer and KARPAS-422 studies (FIG. 10cand FIG. 21a ) and 500 mm³ in the KARPAS-422 intermittent dosing study(FIG. 21b ). Mice were weighed and tumors measured with calipers twiceweekly. For mouse pharmacokinetic studies, tumor and blood samples wereharvested from euthanized mice at the indicated time. Blood and tumorhomogenates were flash frozen and subsequently analyzed by HPLC/MS/MS toevaluate the concentration of GSK126. For pharmacodynamic studies, aportion of each tumor was frozen for H3K27me3/H3 ELISAs or placed inRNAlater (Ambion) for RNA isolation. For peripheral blood analyses,blood was harvested via cardiac puncture from euthanized,immunocompetent CD-1 mice (3 mice/group) on day 18. Blood wasimmediately placed into a Microtainer EDTA tube (BD) and gently mixed byinverting. A complete blood count analysis was conducted using the Advia2120 hematology analyzer (Siemens Medical Solutions) using multi-speciessoftware as per manufacturer's instructions.

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The reference list is cited throughout the text. Each of the referencesis hereby incorporated by reference in its entirety herein, e.g. toprovide additional experimental details. To the extent that thereferences conflict with the claims, embodiments, or definitionsdescribed herein, the instant specification controls.

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TABLE 1 Steady state kinetic parameters for wild type and mutant EZH2enzymes. SAM K_(m) Subtrate K_(m) k_(cat) SAM k_(cat)/K_(m) Substratek_(cat/)K_(m) Enzyme Substrate (μM) (μM) (min⁻¹) (μM⁻¹ min⁻¹)(μM⁻¹min⁻¹) wild type H3K27me0 0.34 ± 0.09 4.23 ± 0.76 0.62 ± 0.04 1.82± 0.44 0.15 ± 0.05 H3K27me1 0.51 ± 0.10 7.44 ± 1.19 0.41 ± 0.03 0.80 ±0.34 0.06 ± 0.03 H3K27me2 0.21 ± 0.06 2.42 ± 0.45 0.016 ± 0.001 0.08 ±0.02 0.007 ± 0.002 nucleosomes 0.10 ± 0.02 0.08 ± 0.01 0.10 ± 0.01 0.99± 0.33 1.22 ± 0.23 A677G H3K27me0 0.34 ± 0.23 4.52 ± 0.99 0.64 ± 0.061.89 ± 0.25 0.14 ± 0.06 H3K27me1 0.89 ± 0.16 10.95 ± 1.45  0.69 ± 0.050.78 ± 0.29 0.06 ± 0.03 H3K27me2 0.95 ± 0.16 5.18 ± 0.84 0.56 ± 0.040.59 ± 0.24 0.11 ± 0.05 nucleosomes 0.20 ± 0.04 0.06 ± 0.01 0.17 ± 0.020.85 ± 0.50 3.04 ± 0.95 Y641N H3K27me0 *—  — — — — H3K27me1 0.64 ± 0.3122.71 ± 6.12  0.42 ± 0.07 0.66 ± 0.23 0.02 ± 0.01 H3K27me2 0.76 ± 0.092.72 ± 0.27 1.00 ± 0.04 1.31 ± 0.48 0.37 ± 0.16 nucleosomes 0.06 ± 0.010.05 ± 0.01 0.073 ± 004  1.28 ± 0.29 1.49 ± 0.35 Y641F H3K27me0 1.19 ±0.39 8.14 ± 2.35 0.08 ± 0.01 0.07 ± 0.03 0.010 ± 0.004 H3K27me1 0.65 ±0.19 15.10 ± 2.62  0.15 ± 0.01 0.23 ± 0.07 0.010 ± 0.005 H3K27me2 0.27 ±0.06 3.93 ± 0.73 0.50 ± 0.04 1.86 ± 0.63 0.13 ± 0.05 nucleosomes 0.08 ±0.02 0.05 ± 0.01 0.07 ± 0.01 0.88 ± 0.25 1.46 ± 0.38 Y641S H3K27me0 — —— — — H3K27me1 0.49 ± 0.14 3.46 ± 0.74 0.21 ± 0.01 0.43 ± 0.09 0.06 ±0.02 H3K27me2 0.49 ± 0.08 2.66 ± 0.39 0.62 ± 0.03 1.26 ± 0.41 0.23 ±0.09 nucleosomes 0.15 ± 0.03 0.07 ± 0.01 0.11 ± 0.01 0.73 ± 0.27 1.55 ±0.42 Y641H H3K27me0 — — — — — H3K27me1 2.14 ± 0.45 6.12 ± 1.44 0.14 ±0.02 0.07 ± 0.04 0.02 ± 0.01 H3K27me2 0.59 ± 0.11 4.05 ± 0.65 0.16 ±0.01 0.26 ± 0.10 0.04 ± 0.02 nucleosomes 0.06 ± 0.01 0.04 ± 0.01  0.04 ±0.002 0.67 ± 0.20 1.00 ± 0.17 Y641C H3K27me0 — — — — — H3K27me1 — — — —— H3K27me2 2.84 ± 0.62 11.99 ± 2.72  0.58 ± 0.07 0.20 ± 0.12 0.05 ± 0.03nucleosomes 0.17 ± 0.04 0.04 ± 0.01  0.03 ± 0.002 0.18 ± 0.05 0.68 ±0.21 *could not obtain value due to low activity

TABLE 2 Clinical profile and mutations identified in primary lymphomasTumor Nucleotide AA Sample ID type Gender Age Change* Change* Mutationtype Zygosity CD563845 DLBCL Male 54 G553G/C D185D/H Non-synonymous SNP(rs2302427) Het CD564749 DLBCL Female 66 G553G/C D185D/H Non-synonymousSNP (rs2302427) Het CD565202 DLBCL Female 65 G553G/C D185D/HNon-synonymous SNP (rs2302427) Het CD564307 MCL Male 52 G553G/C D185D/HNon-synonymous SNP (rs2302427) Het CD564609 WM Female 56 G553G/C D185D/HNon-synonymous SNP (rs2302427) Het CD564308 DLBCL Female 74 G553G/C;D185D/H; Non-synonymous SNP (rs2302427); Non- Het C2045C/G A677A/Gsynonymous missense mutation CD564591 DLBCL Male 34 G553G/C; D185D/H;Non-synonymous SNP (rs2302427); Non- Het; Het A1937A/G Y641Y/Csynonymous missense mutation CD563202 DLBCL Female 57 No Changes NoChanges WT — CD563344 DLBCL Male 74 No Changes No Changes WT — CD563479DLBCL Male 59 No Changes No Changes WT — CD563546 DLBCL Female 75 NoChanges No Changes WT — CD563726 DLBCL Male 44 No Changes No Changes WT— CD563892 DLBCL Male 69 No Changes No Changes WT — CD564332 DLBCL Male62 No Changes No Changes WT — CD564370 DLBCL Male 47 No Changes NoChanges WT — CD564431 DLBCL Male 55 No Changes No Changes WT — CD564439DLBCL Male 58 No Changes No Changes WT — CD564624 DLBCL Male 63 NoChanges No Changes WT — CD564669 DLBCL Male 58 No Changes No Changes WT— CD564960 DLBCL Male 70 No changes No changes WT — CD565057 DLBCL Male65 No changes No changes WT — CD565083 DLBCL Male 58 No changes Nochanges WT — CD565170 DLBCL Male 75 No changes No changes WT — CD565185DLBCL Male 56 No changes No changes WT — CD565213 DLBCL Male 69 Nochanges No changes WT — CD565293 DLBCL Male 66 No changes No changes WT— CD565343 DLBCL Male 61 No changes No changes WT — CD565361 DLBCLFemale 61 No changes No changes WT — CD563565 FL Male 78 No changes Nochanges WT — CD564662 FL Female 58 No changes No changes WT — CD564711FL Female 57 No changes No changes WT — CD565280 FL Male 51 No changesNo changes WT — CD563109 MALT Male 53 No changes No changes WT —CD563451 SMZL Male 55 No changes No changes WT — CD564312 WM Male 63 Nochanges No changes WT — CD563285 DLBCL Male 49 C1477C/T; P488P/S;Non-synonymous missense mutation; Het; Hom G1731G/A P572P Synonymous SNP(rs41277437) CD563860 DLBCL Female 76 G1731G/A P572P Synonymous SNP(rs41277437) Hom CD564738 DLBCL Male 72 G1731G/A P572P Synonymous SNP(rs41277437) Hom CD564672 FL Male 58 A1937A/T Y641Y/F Non-synonymousmissense mutation Het CD564341 FL Male 57 T1936T/C Y641Y/HNon-synonymous missense mutation Het CD564333 DLBCL Male 65 T1936T/AY641Y/N Non-synonymous missense mutation Het Diffuse Large B-cellLymphoma (DLBCL); Splenic marginal zone lymphoma (SMZL); Waldenström'smacroglobulinemia or lymphoplasmacytic lymphoma (WM); Follicularlymphoma (FL); Mantle Cell Lymphoma (MCL); Extra nodal marginal zoneB-cell lymphoma of mucosa assoicated lymphoid tissue (MALT) Wild-type(WT); single nucleotide polymorphism (SNP); Homozygous (Hom);Heterozygous (HET) *Nucleotide/amino acid residue numbering based onNM_001203247

TABLE 3 Primers utilized for sequencing of EZH2 gDNA and cDNA. ReferenceTemplate type Location Primer sequence (5′ → 3′; F: forward; R: reverse)chr7: 148504464- gDNA Exon 2 F: GGTGATCATATTCAGGCTGG 148581441 (hg19R: AAACTTATTGAACTTAGGAGGGG build) Exon 3 F: TTTTGTATTATTTGAATGTGGGAAAR: AAGATGGACACCCTGAGGTC Exon 4 F: ACCCTAAGTAAAAGAAAAGAGAGAAR: GGAAAAGAGTAATACTGCACAGG Exon 5 F: AAATCTGGAGAACTGGGTAAAGACR: TCATGCCCTATATGCTTCATAAAC Exon 6 F: FAGGCTATGCCTGTTTTGTCCR: AAAAGAGAAAGAAGAAACTAAGCCC Exon 7 F: CTGACTGGCATTCCACAGACR: AAGTGTAGTGGCTCATCCGC Exon 8 F: CATCAAAAGTAACACATGGAAACCR: TTGTAATAAATGATAGCACTCTCCA Exon 9 F: TCCATTAATTGACTTTTCCAGTGR: ACCTCCACCAAAGTGCAAAG Exon 10 F: TTCTCTTCCATCAAAATGAGTTTTAR: TCCTCACAACACGAACTTTCAC Exon 11 F: GAGTTGTCCTCATCTTTTCGCR: CCAAGAATTTTCTTTGTTTGGAC Exon 12 F: AAGAATGGTTTGCCTAAATAAGACR: CCTTGCCTGCAGTGTCTATC Exon 13 F: TCTTGGCTTTAACGCATTCCR: CAAATTGGTTTAACATACAGAAGGC Exon 14 F: TGATCGTTTCCATCTCCCTGR: AGGGAGTGCTCCCATGTTC Exon 15 F: GAGAGTCAGTGAGATGCCCAGR: TTTGCCCCAGCTAAATCATC Exon 16 F: TTTGTCCCCAGTCCATTTTCR: TTTCCAATCAAACCCACAGAC Exon 17 F: TTCTGTCAGGCTTGATCACCR: CTCGTTTCTGAACACTCGGC Exon 18- F: TGAAGCTGCTTGATTTATTTGC 19R: AACCTAATTCCCCACTAATGCTC Exon 20 F: TCTCAGCACATGTTGGATGGR: CCCACAGTACTCGAGGTTCC NM_001203247 cDNA 176-193 F: 5′GCGGGACGAAGAATAATC 3′ 538-555 R: 5′ ATAAAATTCTGCTGTAGG 3′ 495-516 F: 5′TGAATGCAGTTGCTTCAGTACC 3′ 956-977 R: 5′ GGGTACATTCAGGAGGAAGTGC 3′886-905 F: 5′ TCCAGATAAGGGCACAGCAG 3′ 1397-1420 R: 5′TTCAGAGGAGCTCGAAGTTTCATC 3′ 1259-1278 F: 5′ GGACGGCTTCCCAATAACAG 3′1788-1808 R: 5′ CTATCACACAAGGGCACGAAC 3′ 1693-1715 F: 5′TGCACACTGCAGAAAGATACAGC 3′ 2223-2242 R: 5′ TTTGTTACCCTTGCGGGTTG 3′1950-1969 F: 5′ TTACTTGTGGAGCCGCTGAC 3′ 2455-2477 R: 5′CTAAGGCAGCTGTTTCAGAGGAG 3′

TABLE 4 Oligonucleotide sequences used for site-directedmutagenesis of EZH2. EZH2 A677G sense5- GAA CAATGA TTT TGT GGT GGATGG AAC CCG CAA GGGTAA CAA AAT TC -3antisense  5- GAATTT TGT TAC CCT TGC GGGTTC CATCCA CCA CAA AAT CAT TGT TC -3 EZH2 Y641N sense antisense EZH2 Y641Fsense 5- GTG CAG AAA AAT GAA TTC ATC TCA GAATTC TGT GGA GAG ATT ATT TCT CAA GAT G -3 antisense5- CAT CTT GAG AAATAATCT CTC CAC AGA ATTCTG AGA TGA ATT CAT TTT TCT GCA C -3 EZH2 Y641S sense5- GAA AAATGA ATT CAT CTC AGA ATC ATGTGG AGA GAT TAT TTC TC -3 antisense5- GAG AAA TAA TCT CTC CAC ATG ATT CTG AGA TGA ATT CAT TTT TC -3EZH2 Y641H sense 5- GAA AAA TGA ATT CAT CTC AGA ACA CTGTGG AGA GAT TAT TTC TC -3 antisense5- GAG AAA TAA TCT CTC CAC AGT GTT CTG AGA TGA ATT CAT TTT TC -3EZH2 Y641C sense 5- GCA GAA AAA TGA ATT CAT CTC AGA ATGCTG TGG AGA GAT TAT TTC TCA AGA TG -3 antisense5- CAT CTT GAG AAA TAA TCT CTC CAC AGCATT CTG AGA TGA ATT CAT TTT TCT GC -3

TABLE 5 H3K27me3 levels, EZH2 mutation status and growth IC50 values ina panel of lymphoma cell lines Ratio of H3K27m3:H3 as Compound A Statusat Y641 pr Cell Line % of Pfeiffer gIC50 (nM) A677 L428 185 3101 Y641SSU-DHL-10 134 324 Y641F Karpass422 106 1316 Y641N WSU-DLCL-2 105 3100Y641F Pfeiffer 100 189 A677G SU-DHL-4 70 13959 Y641S DB 63 9501 Y641NDaudi 55 13371 WT RL 55 4660 Y641N 1A2 55 9264 WT DOHH2 49 6431 WT JM146 6658 WT MJ 46 3278 WT RCK8 40 5563 WT CROAP5 39 6156 WT Jiyoye 392466 WT SU-DHL-5 39 3440 WT Ramos 37 3508 WT BC-3 36 5281 WT Toledo 3421817 WT RPMI-6666 33 3096 WT P3HR1 32 7797 WT Farage 32 4786 WT REC1 322906 WT Hs445 32 1567 WT HDMyZ 30 7207 WT DG75 30 6234 WT Raji 30 6136WT CROAP2 29 11337 WT NAMALWA 28 6223 WT CA46 28 8631 WT U937 27 7898 WTHsSullan 26 3939 WT BC-2 24 2176 WT OCI-LY-19 23 4484 Y641N HT 21 3711WT BC-1 20 8822 WT SKM1 6 6164 Y641C

TABLE 6 GROWTH IC50 VALUES, BASELINE H3K27ME3 LEVELS, MUTATION STATUS,AND TUMOR SUBTYPE FOR LYMPHOMA CELL LINES. AVERAGE GROWTH BASELINEH3K27ME3 CYTOSTATIC/ CELL LINE IC₅₀ ^(a) (nM) LEVEL^(b) EZH2 MUTATIONSTATUS^(c) TUMOR TYPE^(d) SUB-TYPE^(e) t(14:18) STATUS^(f) P53STATUS^(g) CYTOTOXIC^(h) WILL-2 27458  ND^(i) WT B-NHL DLBCL N/A^(j) N/ATOLEDO 13786 34 WT B-NHL DLBCL POSITIVE MUTANT WILL-1 5527 ND D185H^(K)(HET) B-NHL DLBCL N/A N/A SU-DHL-4 4828 70 Y641S (HET); Y661N (HET)B-NHL DLBCL POSITIVE MUTANT RL 4727 55 Y641N; D185H (HET) B-NHL DLBCLN/A N/A U-2940 4558 ND WT B-NHL DLBCL N/A N/A SU-DHL-8 3190 ND WT B-NHLDLBCL NEGATIVE MUTANT U-2932 2935 ND WT B-NHL DLBCL N/A N/A SU-DHL-52299 39 WT B-NHL DLBCL NEGATIVE WT FARAGE 1715 32 D185H (HET) B-NHLDLBCL NEGATIVE MUTANT OCI-LY-19 1019 23 WT B-NHL DLBCL N/A WT DB 861 63Y641N (HET) B-NHL DLBCL POSITIVE MUTANT CYTOSTATIC SU-DHL-6 582 ND Y641N(HET) B-NHL DLBCL POSITIVE MUTANT CYTOSTATIC HT 516 21 WT B-NHL DLBCLNEGATIVE MUTANT CYTOSTATIC SU-DHL-10 448 134  Y641F (HET) B-NHL DLBCLNEGATIVE MUTANT CYTOSTATIC KARPAS-422 232 106  Y641F (HET) B-NHL DLBCLPOSITIVE MUTANT CYTOSTATIC WSU-DLCL2 134 105  Y641F (HET) B-NHL DLBCLN/A MUTANT CYTOSTATIC PFEIFFER 28 100  A677G (HET) B-NHL DLBCL POSITIVEWT CYTOSTATIC RAJI 7865 30 WT B-NHL BURKITT N/A N/A CA46 6585 28 D185H(HET) B-NHL BURKITT N/A N/A DG-75 3254 30 WT B-NHL BURKITT N/A N/AP3HR-1 3207 32 WT B-NHL BURKITT N/A N/A HS-SULTAN 2275 26 WT B-NHLBURKITT N/A N/A DAUDI 1265 55 WT B-NHL BURKITT N/A N/A JIYOYE 232 39 WTB-NHL BURKITT N/A N/A CYTOSTATIC BC-1 8292 20 WT B-NHL AIDS-BCBL N/A N/ABC-2 4762 24 WT B-NHL AIDS-BCBL N/A N/A BC-3 2217 36 D185H (HET) B-NHLPEL, AIDS-BCBL N/A N/A CRO-AP2 1643 ND WT B-NHL PEL, AIDS-BCBL N/A N/AWSU-FSCCL 11966 ND D185H (HET) B-NHL FL N/A N/A SC-1 3727 ND D185H (HET)B-NHL FL N/A WT WSU-NHL 3537 ND D185H (HET) B-NHL FL POSITIVE MUTANTNU-DUL-1 17060 ND WT B-NHL N/A N/A MUTANT MC116 10168 39 WT B-NHL N/AN/A MUTANT RI-1 7656 ND WT B-NHL MCL N/A MUTANT MINO 7340 ND D185H (HET)B-NHL N/A N/A N/A U-698-M 5599 ND WT B-NHL N/A N/A MUTANT MHH-PREB-15300 33 WT B-NHL N/A N/A MUTANT KARPAS-1106P 4536 ND E744A (HET) B-NHLMLBCL N/A WT RC-K8 4528 40 D185H (HET) B-NHL N/A N/A WT CI-1 4282 NDD185H (HET) B-NHL N/A N/A N/A SU-DHL-16 3282 ND D185H (HET) B-NHL N/AN/A MUTANT HD-MY-Z 10717 30 WT HODGKIN'S N/A N/A N/A L-428 4704 185 Y641S (HET) HODGKIN'S N/A N/A N/A Hs 445 3528 32 WT HODGKIN'S N/A N/AN/A RPMI-6666 1429 33 WT HODGKIN'S N/A N/A N/A ^(a)GROWTH IC50 VALUESREPRESENT THE AVERAGE OF AT LEAST 2 INDEPENDENT REPLICATE EXPERIMENTS^(b)BASELINE H3K27ME3 LEVELS ARE REPRESENTED RELATIVE TO THE LEVELOBSERVED IN THE PFEIFFER CELL LINE. ^(c)AMINO ACID RESIDUE NUMBER BASEDON NP_001190176. ^(d)B-NHL, B-CELL NON-HODGKIN LYMPHOMA. HODGKIN'S,HODGKIN'S LYMPHOMA. ^(e)DLBCL, DIFFUSE LARGE B-CELL LYMPHOMA. BURKITT,BURKITT LYMPHOMA. PEL, PRIMARY EFFUSION LYMPHOMA. AIDS-BCBL, ACQUIREDIMMUNODEFICIENCY SYNDROME BODY CAVITY-BASED LYMPHOMA. MLBCL, MEDIASTINALLARGE B CELL LYMPHOMA. MCL, MANTLE CELL LYMPHOMA. FL, FOLLICULARLYMPHOMA. N/A, NOT AVAILABLE. ^(f)DENG ET AL., CANCER CELL 12: 171-185(2007). ^(g)DORNAN ET AL., BLOOD 114: 2721-29 (2009). ^(h)DOSE-RESPONSECURVES FROM CELL LINES WITH GROWTH IC₅₀ VALUES < 1 μM WERE EVALUATED FOREVIDENCE OF CYTOSTASIS OR CYTOTOXICITY (VALUES BELOW T

). ^(i)NOT DETERMINED. ^(j)NOT AVAILABLE. ^(K)D185h IS A KNOWN SNP(RS2302427).

indicates data missing or illegible when filed

TABLE 7 Common up-regulated genes in at least 4 out of 5 sensitivemutant cell lines^(a) WSU- KARPAS- SU- SU- OCI- SU- KARPAS- WSU-Pfeiffer DLCL2 422 DHL-10 HT DHL-6 DB LY-19 DHL-4 Toledo Pfeiffer 422DLCL2 Entrez Fold Fold Fold Fold Fold Fold Fold Fold Fold Fold H3K27me3H3K27me3 H3K27me3 Probeset ID Symbol Description Gene Unigene ChangeChange Change Change Change Change change Change Change Change MarkedMarked Marked 201310_s_at C5orf13 chromo- 9315 Hs.483067 2.68 2.04 4.652.39 1.55 1.45 2.24 1.06 1.01 1.09 No No No some 5 open reading frame 13201403_s_at MGST3 microsomal 4259 Hs.191734 2.14 3.30 2.49 2.68 1.641.93 1.41 1.28 1.34 1.05 Yes Yes Yes glutathione S-Trans- ferase 3201760_s_at WSB2 WD repeat 55884 Hs.719911 1.54 4.89 5.83 2.43 1.38 2.580.84 0.95 2.24 1.07 Yes Yes Yes and SOCS box- containing 2 203504_s_atABCA1 ATP-binding 19 Hs.429294 3.66 1.56 11.38 2.73 1.81 2.59 2.56 0.861.03 0.97 Yes Yes Yes cassette, sub-family A (ABC1), member 1 203505_atABCA1 ATP-binding 19 Hs.429294 3.26 1.48 11.53 2.50 3.79 2.37 2.77 1.061.02 0.88 Yes Yes Yes cassette, sub-family A (ABC1), member 1206857_s_at FKBP1B FK506 2281 Hs.709461 2.39 2.90 11.45 4.52 2.79 2.641.69 3.37 2.33 1.34 Yes Yes Yes binding protein 1B, 12.6 kDa 207071_s_atACO1

tase 1, 48 Hs.567229 2.02 3.68 0.82 2.67 2.08 2.01 1.13 1.44 1.70 1.37Yes Yes Yes soluble 208999_at SEPT8 sep

 8 23176 Hs.522057 0.87 2.43 4.42 21.93 1.71 4.81 1.44 1.75 2.45 1.10 NoNo No 209459_s_at ABAT 4-amino- 18 Hs.336768 6.66 4.02 18.93 7.04 2.745.39 2.84 2.11 1.48 1.11 Yes Yes Yes butyrate amino- transferase209504_s_at PLEKHB1 pleckstrin 58473 Hs.445489 2.63 6.62 4.45 2.88 1.792.92 1.95 1.68 1.53 1.00 Yes Yes Yes homology domain containing, familyB (evectins) member 1 209605_at TST thiosulfate 7263 Hs.474783 2.52 4.523.20 2.84 1.90 3.45 1.05 1.10 1.83 1.10 Yes Yes Yes sulfurtrans- ferase(rhodanese) 20

995_s_at TCL1A T-cell 8115 Hs.2484 2.47 37.87 9.18 3.79 1.17 126 1.101.04 1.16 1.04 Yes Yes Yes leukemia/ lym

1A 210778_s_at MXD4 MAX 10608 Hs.655020 3.50 3.48 2.09 2.68 2.29 3.441.11 1.49 0.91 0.65 Yes Yes Yes

protein 4 211474_s_at SERPIN

6 serpin 5269 — 2.02 2.02 1.11 5.86 2.87 2.95 1.12 1.61 2.13 1.4

Yes Yes Yes peptidase inhibitor, clade B (ovalbumin), member 6 212778_atPACS2 phospho- 23241 Hs.525626 1.63 2.58 3.01 2.33 1.59 3.15 1.26 1.731.28 1.09 Yes Yes Yes furin acidic cluster sorting protein 2 213309_atPLCL2 phospho- 23228 Hs.202010 3.21 2.17 1.95 3.01 1.82 2.03 1.05 1.162.07 0.92 Yes Yes Yes lipase C- like 2 218436_at CNR1 cannabinoid 1268Hs.709067 13.88 3.69 2.02 4.00 1.76 2.03 1.10 1.11 1.10 1.19 Yes Yes Yesreceptor 1 (brain) 213901_x_at RBM9 RNA 23543 Hs.282998 9.09 2.35 3.943.31 1.03 2.85 1.14 4.31 1.21 1.12 Yes Yes Yes binding

protein 9 218175_at CCDC92 coiled-coil 80212 Hs.114111 2.67 2.72 4.914.05 2.73 2.38 1.80 1.45 1.42 1.07 Yes Yes Yes domain containing 92218346_s_at SESN1

 1 27244 Hs.591336 2.29 6.75 1.62 4.84 2.41 2.73 1.58 1.50 1.95 0.99 YesYes Yes 218773_s_at MSRB2 methionine 22921 Hs.461420 2.53 4.68 4.86 4.691.72 6.19 1.69 1.43 1.74 1.10 No Yes Yes sul

xide reductase B2 219841_at AICDA activation- 57379 Hs.149342 2.43 3.1618.71 1.53 1.26 2.34 3.56 1.87 1.17 3.35 Yes No Yes induced c

ne deaminase 221676_s_at CORO1C

nin, 23603 Hs.330384 2.13 2.56 2.87 2.88 1.77 1.62 1.49 1.84 1.06 1.02Yes Yes Yes actin binding protein, 1C 221773_at ELK3 ELK3, 2004 Hs.46

523 3.88 2.29 1.69 2.36 1.32 3.01 1.81 0.92 0.93 1.09 Yes Yes YesETS-domain protein (SRF accessory protein 2) 222409_at CORO1C coronin,23603 Hs.330384 2.02 2.31 3.08 2.47 1.58 1.72 1.65 1.51 1.02 1.02 YesYes Yes acting binding protein, 1C 224499_s_at AICDA activation- 57379Hs.149342 2.53 3.04 27.88 0.47 1.27 2.46 1.15 0.65 1.22 0.98 Yes No Yesinduced cytidine dea

nase 225123_at SESN3 sestrin 3 143686 Hs.659934 4.61 4.26 5.71 3.41 3.764.87 6.09 1.78 0.98 1.06 Yes No Yes 226272_at RCAN3 RCAN family 11123Hs.656799 2.87 3.01 3.00 2.45 1.96 1.44 2.10 1.49 1.21 1.04 Yes Yes Yesmember 3 227001_at NIPAL2 NIPA-l

79815 Hs.309489 2.73 2.

6 2.90 2.90 1.13 2.30 1.20 1.24 1.33 1.02 Yes Yes Yes domain containing2 227478_at SETBP1 SET binding 26040 Hs.435458 2.76 5.63 3.32 2.04 2.382.11 1.83 0.37 1.73 1.53 Yes Yes Yes protein 1 228377_at KLHL14 ke

-like 14 57565 Hs.446164 1.62 2.46 5.75 4.71 1.78 2.93 0.96 1.37 1.301.09 No Yes Yes (Drosophila) 235352_at — — — — 3.33 2.24 6.47 2.16 1.631.99 1.18 1.37 1.49 0.94 242448_at

AP4K3 mitogen- 8491 Hs.655750 2.93 3.29 5.67 2.04 2.53 2.47 1.66 1.121.89 0.78 Yes Yes Yes activated protein kinase kinase kinase kinase 3244166_at APLN ap

n 8862 Hs.303084 1.31 5.52 5.33 5.38 1.36 2.12 1.48 1.12 1.56 0.73 NoYes Yes 39318_at TCL1A T-cell 8115 Hs.2484 3.11 31.65 10.47 4.41 1.101.34 1.10 1.03 1.20 1.03 Yes Yes Yes leukemia/ lymphoma 1A ^(a)Pfeiffer,KARPAS-422, WSU-DLCL2, SU-DHL-10, and SU-DHL-6.

indicates data missing or illegible when filed

What is claimed is:
 1. A kit for the treatment of cancer comprising akit for determining one or more of the following in a sample from ahuman: a. the presence or absence of a mutation at the alanine 677(A677) residue in EZH2 Enhancer of Zeste Homolog 2 (EZH2) in a samplefrom said human; or b. the presence or absence of a mutation at thetyrosine 641 (Y641) residue in EZH2; or c. the presence or absence of anincreased level of H3K27me3 as compared to a control, and a means fordetermining one or more of a, b, and c.
 2. The kit of claim 1, whereinsaid means is selected from the group consisting of primers, probes, andantibodies.